CN114702585A

CN114702585A - anti-CD 22 monoclonal antibody 4F6, and preparation method and application thereof

Info

Publication number: CN114702585A
Application number: CN202111420965.2A
Authority: CN
Inventors: 万晓春; 李俊鑫; 刘茂玄
Original assignee: Shenzhen Bindebiotech Co ltd
Current assignee: Shenzhen Bindebiotech Co ltd
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-07-05
Anticipated expiration: 2041-11-26
Also published as: CN114702585B

Abstract

The invention relates to a monoclonal antibody 4F6 resisting CD22, a preparation method and application thereof, and particularly discloses an anti-4F 6 antibody or an antigen binding fragment thereof, which has three heavy chain Complementarity Determining Regions (CDRs) shown as SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, and has three light chain complementarity determining regions shown as SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28. Also disclosed is a nucleotide sequence characterized in that: encoding an anti-CD 22 monoclonal antibody or antigen-binding fragment thereof as described previously; and the use of the monoclonal antibody as a detection reagent. The antibody has the advantages of high sensitivity, good specificity and the like.

Description

anti-CD 22 monoclonal antibody 4F6, and preparation method and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to a monoclonal antibody for resisting CD22, a preparation method and application thereof.

Background

CD22, entitled Cluster of differentiation-22 (English: cluster of differentiation-22), is a transmembrane receptor on the surface of mature B cells and belongs to the SIGLEC family. Its main function is to avoid the over-activation of immune system and reduce the risk of autoimmune diseases. CD22 is a co-receptor for B cell receptors, which is itself phosphorylated when linked to an antigen bound to the surface of an antigen B cell receptor, thereby activating certain phosphatases to suppress the immune response. CD22 may also be targeted as an anti-cancer drug, and the national institutes of health in the united states is testing an immunotoxin named BL22 (a monoclonal antibody against CD 22), which binds to CD22 and can enter cells to achieve a cell killing effect, possibly contributing to the treatment of certain types of leukemia. Another monoclonal antibody drug, Epratuzumab, directed against CD22 is also being tested and may be useful in the treatment of leukemia and systemic lupus erythematosus. Meanwhile, the anti-CD 22 antibody can be combined with molecules such as toxin and radioactive isotope, and the possibility is provided for targeted chemotherapy or radiotherapy of tumor cells. Due to the high specificity of antibody antigen recognition, monoclonal antibodies targeting CD22 will greatly reduce the side effects of therapy.

The position of antibody products as the most widely used tool in the field of biological research is undoubted. The specificity and range of application of antibodies has long plagued the antibody industry and is a significant crisis in the entire field of biological research. The poor quality of the antibody product directly leads to errors in experimental results, and the results of the study cannot be repeated and reproduced. The progress of the research project is often delayed by the problems associated with antibodies, and the resulting losses are also surprising. According to 2011 statistics, an average of $ 3.5 billion per year is wasted on these ineffective antibodies in the U.S. alone. In the world, 8 hundred million America yuan is wasted in each year, and accounts for 50% of the total cost of the antibodies in the global scientific research.

Disclosure of Invention

In order to overcome the problems, the invention provides a monoclonal antibody 4F6 of anti-CD 22 protein, which can be applied to experiments such as immune imprinting, flow cytometry, ELISA and the like and is a powerful tool for researching the function of CD 22.

One aspect of the present invention provides an isolated antibody against CD22 protein or an antigen binding fragment thereof, having three heavy chain Complementarity Determining Regions (CDRs) as set forth in

SEQ ID NO

2, 3, and 4, and having three light chain complementarity determining regions as set forth in SEQ ID NO 10, 11, and 12.

Another aspect of the invention provides an isolated anti-CD 22 protein antibody or antigen-binding fragment thereof having an amino acid sequence as set forth in SEQ ID No:1, and the light chain variable region shown in SEQ ID No. 9.

In a further aspect, the present invention provides a nucleotide sequence which is characterized in that: which encodes a monoclonal antibody or antigen-binding fragment thereof against CD22 protein as described previously.

In the technical scheme of the invention, the antibody is a monoclonal antibody.

In a further aspect of the present invention, there is provided a recombinant vector characterized in that: comprising the nucleotide sequence described above.

In a further aspect of the invention, there is provided a host cell characterised in that: comprising the aforementioned vector or vectors, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of yeast cells, mammalian cells or other cells suitable for the production of antibodies or antigen binding fragments thereof.

In a further aspect of the invention, there is provided a kit comprising an antibody or antigen-binding fragment thereof as described above.

In yet another aspect, the invention provides a detection reagent comprising an antibody or antigen-binding fragment thereof as described above.

In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above as a detection reagent for: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), Immunohistochemical (IHC) assay, or immuno-PCR.

In the above-mentioned immunological assay, the antibody or antigen-binding fragment thereof may be attached alone or in combination with a conjugate such as horseradish peroxidase (HRP), Alkaline Phosphatase (AP), Biotin (Biotin), Fluorescein Isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose, by coupling via a chemical bond, electrostatic adsorption or hydrophilic-hydrophobic adsorption.

In the technical scheme of the invention, the detection reagent can be used for detection for non-diagnostic treatment purposes.

In a further aspect, the invention provides the use of the above antibody or antigen-binding fragment thereof as a reagent for the in vitro isolation or purification of CD22 protein.

In the present invention, the above-mentioned antibody or antigen-binding fragment thereof is produced by a hybridoma method.

In the present invention, the nucleotide and amino acid sequences of the heavy and light chain variable regions of the antibody or antigen-binding fragment thereof are shown below.

Heavy chain variable region amino acid sequence:

EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGITIYN QKFKGKATLTIDKSSSTAYMELRSLTSEDTAVYYCARDRYDRGYALDYWGQGTSVTVSS

SEQ ID NO.1

wherein, the heavy chain variable region CDR1 amino acid sequence is: DYNMD SEQ ID NO.2

The heavy chain variable region CDR2 amino acid sequence is: DIDPNNGITIYNQKFKG SEQ ID NO.3

The heavy chain variable region CDR3 amino acid sequence is: DRYDRGYALDY SEQ ID NO.4

The amino acid sequence of heavy chain variable region FR1 is: EVLLQQSGPELVKPGASVKIPCKASGYTFT SEQ ID NO.5

The amino acid sequence of heavy chain variable region FR2 is: WVKQSHGKSLEWIG SEQ ID NO.6

The amino acid sequence of FR3 in the heavy chain variable region is: KATLTIDKSSSTAYMELRSLTSEDTAVYYCAR SEQ ID NO.7

The amino acid sequence of heavy chain variable region FR4 is: WGQGTSVTVSS SEQ ID NO.8

Light chain variable region amino acid sequence: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWHQQKTGQSPKLLVYFASIRD SGVPDRFIGSGSGTDFTLTINSVQAEDLADYFCQQHYSTPFTFGSGTKLEIK SEQ ID NO.9

Wherein the light chain variable region CDR1 amino acid sequence is: KSSQSLLNSSNQKNYLA SEQ ID NO.10

The light chain variable region CDR2 amino acid sequence is: FASIRDS SEQ ID NO.11

The light chain variable region CDR3 amino acid sequence is: QQHYSTPFT SEQ ID NO.12

The amino acid sequence of the light chain variable region FR1 is: DIVMTQSPSSLAMSVGQKVTMSC SEQ ID NO.13

The amino acid sequence of the light chain variable region FR2 is: WHQQKTGQSPKLLVY SEQ ID NO.14

The amino acid sequence of the light chain variable region FR3 is: GVPDRFIGSGSGTDFTLTINSVQAEDLADYFC

SEQ ID NO.15

The amino acid sequence of the light chain variable region FR4 is: FGSGTKLEIK SEQ ID NO.16

The heavy chain variable region gene has a total length of 360bp and 120 coded amino acid residues. The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 17, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 4.

The total length of the light chain variable region gene sequence is 339bp, and 113 coded amino acid residues are provided. The light chain variable region nucleotide sequence is shown as SEQ ID NO. 25, the light chain variable region amino acid sequence is shown as SEQ ID NO.9, the light chain CDR1 amino acid sequence is shown as SEQ ID NO.10, the light chain CDR2 amino acid sequence is shown as SEQ ID NO.11, and the light chain CDR3 amino acid sequence is shown as SEQ ID NO. 12.

Advantageous effects

The invention provides a preparation method, identification and application of a monoclonal antibody of anti-CD 22 protein, and the monoclonal antibody has the advantages of high sensitivity, good specificity and the like, and is suitable for different detection methods. The monoclonal antibody provided by the invention can be widely used for detecting CD22 protein by different means such as Western blot, ELISA, flowCytometry and the like, and provides a basis for researching the function of human CD22 protein.

Drawings

FIG. 1 is a graph showing the affinity results of the 4F6 antibody for CD22 protein.

FIG. 2 shows the results of 4F6 in example 4 applied to flow cytometry.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments of the present invention are described in detail below, but the present invention is not to be construed as limiting the implementable range of the present invention.

EXAMPLE 1 production of CD22 monoclonal antibody

The hybridoma method (first proposed by Kohler et al, Nature,256:495 (1975)) was used. A female BALB/c mouse (6 weeks old) is immunized by using a CD22 protein antigen (purchased from Chinesia, Chinesia) and emulsified antigens are used for the first immunization by using Freund's complete adjuvant, and the emulsified antigens are used for the second immunization and injected at 5-6 subcutaneous points, wherein the amount of the antigens injected by each mouse is-100 mu g. 10 days after the 3 rd immunization, a small amount of blood is collected from the tail of the mouse and subjected to serum titer ELISA detection, and the 4 th immunization is performed by selecting the mouse with high antibody titer (>1:100000), and 100 mu g of antigen protein is injected into each mouse through the abdominal cavity. 3-5 days after 4 th immunization, the spleen cells of the killed mice are fused with SP2/0 cells, and the stable hybridoma cells are obtained through HAT culture medium culture. Screening hybridoma capable of secreting CD22 antibody by ELISA method, subcloning by limiting dilution method, screening monoclonal hybridoma capable of secreting CD22 antibody by ELISA method, expanding culture step by step, freezing with liquid nitrogen and preserving seed.

Preparing an ascites antibody: female BALB/c mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, each mouse was injected with 0.5mL, 3-5 days later, the mice were intraperitoneally injected with hybridoma cells in logarithmic phase, and each mouse was injected with 1-5 × 10⁵Individual cells (0.5mL), mice were sacrificed 11 days after injection of hybridoma cells to obtain ascites fluid. Centrifuging at 3000rpm and 4 deg.C for 10min, removing precipitate, diluting ascites with 10 times volume of 1 XPB solution, mixing, and filtering with 0.45 μm filter membrane. Ascites fluid was affinity-purified by Protein G (Protein G Sepharose 4Fast Flow, GE Healthcare) to obtain a purified CD22 antibody Protein, and the antibody concentration was measured by BCA method. The purified antibody was run on SDS-PAGE (loading 5.4. mu.g) and stained with Coomassie Brilliant blue.

Example 2 antibody Gene sequencing of CD22 monoclonal antibody hybridoma cells

Harvesting monoclonal antibody hybridoma cells in logarithmic growth phase, carrying out TRIZOL lysis to carry out RNA extraction, carrying out reverse transcription to obtain cDNA, amplifying and obtaining heavy chain and light chain variable regions, removing non-functional VK genes, cloning to a pMD18-T vector, sequencing, comparing sequencing results by using an IMGT/V-QUEST database, and further analyzing.

The amino acid sequence of the heavy chain of monoclonal antibody 4F6 is shown below: EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGITIYN QKFKGKATLTIDKSSSTAYMELRSLTSEDTAVYYCARDRYDRGYALDYWGQGTSVTVSS

SEQ ID No. 1; wherein the content of the first and second substances,

the heavy chain CDR1 amino acid sequence is: DYNMD SEQ ID NO.2

The heavy chain CDR2 amino acid sequence is: DIDPNNGITIYNQKFKG SEQ ID NO.3

The heavy chain CDR3 amino acid sequence is: DRYDRGYALDY SEQ ID NO.4

The amino acid sequence of heavy chain FR1 is: EVLLQQSGPELVKPGASVKIPCKASGYTFT SEQ ID NO.5

The amino acid sequence of heavy chain FR2 is: WVKQSHGKSLEWIG SEQ ID NO.6

The amino acid sequence of heavy chain FR3 is: KATLTIDKSSSTAYMELRSLTSEDTAVYYCAR SEQ ID NO.7

The amino acid sequence of heavy chain FR4 is: WGQGTSVTVSS SEQ ID NO.8

The amino acid sequence of the light chain of monoclonal antibody 4F6 is shown below: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWHQQKTGQSPKLLVYFASIRD SGVPDRFIGSGSGTDFTLTINSVQAEDLADYFCQQHYSTPFTFGSGTKLEIK SEQ ID NO.9

Wherein the light chain CDR1 amino acid sequence is: KSSQSLLNSSNQKNYLA SEQ ID NO.10

The light chain CDR2 amino acid sequence is: FASIRDS SEQ ID NO.11

The light chain CDR3 amino acid sequence is: QQHYSTPFT SEQ ID NO.12

The amino acid sequence of light chain FR1 is: DIVMTQSPSSLAMSVGQKVTMSC SEQ ID NO.13

The light chain FR2 amino acid sequence is: WHQQKTGQSPKLLVY SEQ ID NO.14

The light chain FR3 amino acid sequence is: GVPDRFIGSGSGTDFTLTINSVQAEDLADYFC SEQ ID NO.15

The light chain FR4 amino acid sequence is: FGSGTKLEIK SEQ ID NO.16

Example 3 affinity assay for CD22 monoclonal antibody

The sensor was immobilized using 10 μ g/mL CD22 protein, different concentrations of 4F6 antibody (500nM, 250nM, 125nM, 62.5nM, 31.25nM, 10.41nM, 5.21nM) were formulated using SD buffer (PBS + 0.02% Tween 20+ 0.1% BSA) as mobile phase, and affinity detection was performed using an intermolecular interactor (OCTET K2, PALL life science) programmed as: baseline 240s, Loading 360s, Baseline 2180 s, Association 480s, using AHC (Anti-hIgG Fc Capture) sensors. The results are shown in fig. 1, and the affinity KD of the 4F6 antibody to CD22 is 3.14 × 10^-9M。

1. EXAMPLE 4 use of the CD22 monoclonal antibody

4.1 flow assay application of CD22 monoclonal antibody

1) Collecting acute B lymphocyte leukemia (B-ALL) cells, blowing off, counting, and packaging into 5 centrifuge tubes (3 × 10 per tube)⁵Each cell was centrifuged at 500g for 5min, and the supernatant was removed, and 1mL of PBS was added thereto, and centrifuged at 500g for 5min, and the supernatant was removed.

2) Reagents were added as follows:

placing on ice, incubating for 30 min.

3) 1mL of PBS + 5% FBS was added, the mixture was centrifuged at 500g for 5min, the supernatant was removed, 100. mu.L of DMEM culture solution + 1. mu.L of APC coat anti-mouse lgG (minor x-reactivity) antibody (biolegend) was added, and the mixture was incubated on ice for 20 min.

4) 1mL of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

5) Add 300. mu.L PBS + 5% FBS to each tube, resuspend, and detect using Beckman Cytoflex CM.

As shown in FIG. 2, the 4F6 antibody can be combined with CD22 protein on the surface of B-ALL cells, and is applied to flow cytometry detection, and the detection sensitivity is superior to that of a commercial positive control.

4.2 ELISA detection application of CD22 monoclonal antibody

100ng of CD22 protein is coated on each well of the ELISA plate, the plate is washed at 4 ℃ overnight, and after the plate is sealed and washed, 4F6 antibody with the concentration of 1 mu g/mL is added, each well is 100 mu L, and the plate is incubated for 1h at 37 ℃. PBST wash plate 5 times, add 1:5000 diluted HRP-goat anti mouse IgG antibody, 37 degrees C were incubated for 45 min. And (3) washing the PBST for 5 times, adding 100 mu L of TMB into each hole, reacting for 3min at room temperature in a dark place, adding 50 mu L of stop solution into each hole, stopping reaction, and measuring OD450 by using an enzyme-linked immunosorbent assay. The results show that 4F6 can be used for ELISA detection applications.

SEQUENCE LISTING

<110> Shenzhen Binje Biotechnology Limited

<120> monoclonal antibody 4F6 for resisting CD22, preparation method and application thereof

<130> CP121010746C

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Claims

1. An isolated anti-CD 22 antibody or antigen-binding fragment thereof, characterized in that: it has three heavy chain complementarity determining regions shown as SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, and has three light chain complementarity determining regions shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12;

wherein, the heavy chain complementarity determining region CDR1 is shown in SEQ ID NO. 2;

heavy chain complementarity determining region CDR2 is shown in SEQ ID NO 3;

heavy chain complementarity determining region CDR3 is shown in SEQ ID NO. 4;

light chain complementarity determining region CDR1 is shown in SEQ ID NO 10;

light chain complementarity determining region CDR2 is shown in SEQ ID NO: 11;

light chain complementarity determining region CDR3 is shown in SEQ ID NO 12.

2. An isolated anti-CD 22 antibody or antigen-binding fragment thereof, characterized in that: it has the heavy chain variable region as shown in SEQ ID No.1 and the light chain variable region as shown in SEQ ID No. 9.

3. A nucleotide, characterized in that: encoding the anti-CD 22 antibody or antigen-binding fragment thereof of claim 1 or 2.

4. A recombinant vector, characterized in that: comprising the nucleotide sequence of claim 3.

5. A host cell, characterized in that: comprising the recombinant vector of claim 4;

preferably, the host cell is prokaryotic or eukaryotic;

preferably, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the production of an antibody or antigen-binding fragment thereof.

6. A kit, characterized in that: the kit comprises the antibody or antigen-binding fragment thereof of claim 1 or 2.

7. A detection reagent, characterized in that: the detection reagent comprises the antibody or antigen-binding fragment thereof of claim 1 or 2;

preferably, the detection reagent is used for enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical detection or immuno-PCR.

8. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2 in the preparation of a reagent for detecting CD 22.

9. The use according to claim 8, the agent being an agent for: enzyme linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical assay or immuno-PCR.

10. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2 in the preparation of a reagent for the in vitro isolation or purification of CD 22.