KR101650046B1 - Antibody to specific primates CD19 and a hybridoma producing the same - Google Patents

Abstract

The present invention relates to a hybridoma producing an anti-primate CD19 antibody which recognizes a primate CD19 antigen; to an anti-primate CD19 antibody produced by the hybridoma; and to a detection method of a primate CD19 expressing cell using the anti-primate CD19 antibody. According to the present invention, the anti-primate CD19 antibody has high affinity for a primate CD19 antigen, and thus can be applied to detect a primate CD19 expressing cell. Furthermore, the anti-primate CD19 antibody can be applied to analyze subtypes of a B cell, and thus can be practically used to research a B cell-derived autoimmune disease, isotransplantation, and xenotransplantation. A hybridoma (KCLRF-BP-00337) produces an anti-Rhesus monkey CD19 antibody which recognizes a Rhesus monkey CD19 antigen. A detection method of a Rhesus monkey CD19 expressing cell comprises a step of inducing contact between a Rhesus monkey CD19 antigen and a clinical specimen to perform an antigen-antibody reaction.

Description

Antibody specific for primate CD19 and a fusion cell line producing the antibody and a hybridoma producing the same [

The present invention relates to a fusion cell line that produces an anti-primate CD19 antibody that recognizes a primate CD19 antigen, an anti-primate CD19 antibody produced by the fusion cell line, and a method of detecting a primate CD19 expressing cell using the antibody.

Non-human primates are divided into apes and monkeys. Among the monkeys, approximately 30 species are being used as experimental animals. The monkeys widely used in the world are Macaca genus monkeys such as Rhesus monkey, Cynomolgus monkey, (Baboon) monkeys and common marmosets. Numerous studies have been reported centering on Lethus monkeys. Recently, there has been a boom in research using primates, such as research on diseases such as AIDS, brain diseases and gene therapy using monkeys, and research on artificial organs such as transplanted organs (Biotechnology Policy Research Center, 27-62009-09 -01, Biozine, 2004-06-17). However, in order to succeed, it is important to establish understanding and analysis method of monkey immunity system. To do this, production of monkey-specific antibodies, which are essential for the change and function analysis of each immune cell, should be preceded. However, there are no commercially available antibodies that recognize the Rhesus monkey CD19 antigen. Since there is no anti-human CD19 antibody that cross-reacts with monkey CD19 antibody, it is urgent to develop an antibody specifically recognizing monkey CD19 antigen.

In order to solve the above problems, the present inventors produced a fusion cell line producing an anti-primate CD19 antibody. The anti-primate CD19 antibody generated from the fusion cell line has high affinity for the primate-specific CD19 antigen, and thus is useful for detecting primate CD19 expressing cells, thus completing the present invention.

It is an object of the present invention to provide a fusion cell line which produces an anti-primate CD19 antibody recognizing primate CD19 antigen.

It is also an object of the present invention to provide an anti-primate CD19 antibody produced by said fusion cell line.

It is another object of the present invention to provide a method for detecting a primate CD19-expressing cell, comprising the step of contacting an antibody with a sample of a specimen to perform an antigen-antibody reaction.

In order to solve the above problems, the present invention provides a fusion cell line (Accession No. KCLRF-BP-00337) which produces an anti-primate CD19 antibody recognizing a primate CD19 antigen.

The present invention also provides an anti-primate CD19 antibody produced by the fusion cell line.

Also, the present invention provides a method for detecting a primate CD19-expressing cell, comprising contacting the antibody with a sample of a specimen to perform an antigen-antibody reaction.

The anti-primate CD19 antibody according to the present invention has high affinity for the primate CD19 antigen and can be applied to the detection of primate CD19 expressing cells. Furthermore, it can be applied to the subtype analysis of B cells, and thus can be usefully used for B cell-derived autoimmune diseases, allogeneic and xenograft studies.

Brief Description of the Drawings Figure 1 is a diagram showing the nucleotide sequence and amino acid sequence of CD19 cDNA derived from Rhesus monkey.
2 is a schematic diagram of a CD19-hIg protein expression vector derived from Lethus monkey.
Figure 3 shows the result of Western blot analysis of lysate CD19-hIg protein lysate.
4 is a schematic diagram of a CD19 expression vector in the membrane-bound form of Lysus monkey.
FIG. 5 is a graph showing the results of screening for CD19-CHO cells derived from Lethus monkey using a high concentration anti-human CD19 antibody and a flow cytometer.
FIG. 6 shows eleven clone names (parentheses indicate preliminary clones) as candidates for a fusion cell line producing anti-primate CD19 antibody obtained through primary and secondary screening.
FIG. 7 shows the results of confirming reactivity of the finally selected antibodies (RM6H9 and RM6D9) with the commercialized anti-monkey CD19 (primary antibody: RM9H9 or RM secondary antibody: FL4-H, anti-mouse Ig-APC).
Figure 8 confirms the affinity difference for the detection of monkey CD20 + B cells with the RM6H9 antibody (anti-primate CD19 antibody, A) and the commercialized anti-monkey CD19 antibody (J4.119 clone, B) , B), and monkey B cell specificity (C, D, E).
FIG. 9 is a graph showing the results of detection of anti-human B cells by the antibody of RM6H9
FIG. 10 is a graph showing the results of analysis of a monkey B cell subtype using an antibody of RM6H9.

The present invention provides a fusion cell line that produces an anti-primate CD19 antibody that recognizes a primate CD19 antigen.

The term "primate" in the present invention is preferably a higher primate, more preferably a human or a monkey.

As used herein, the term "antibody" is an antibody that specifically binds to a primate CD19 antigen, including its complete antibody form as well as its antigen binding fragment.

In the present invention, the term "complete antibody" refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being linked by a disulfide bond with a heavy chain.

The term "antigen-binding fragment of an antibody molecule" in the present invention means a fragment having an antigen-binding function and includes Fab, F (ab ') 2, F (ab') 2 and Fv. The Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. The F (ab ') differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 is generated while the cysteine residue of the hinge region of F (ab') forms a disulfide bond. The Fv refers to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Such antibody fragments can be obtained using protein hydrolyzing enzymes according to methods known in the art. For example, Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by digesting with pepsin. The antibody fragments can also be produced by recombinant DNA technology.

In the present invention, the term "variable region" means a portion of an antibody molecule that exhibits a large number of mutations in sequence while performing a function of specifically binding to an antigen. CDR1, CDR2 and CDR3, which are complementarity determining regions (CDRs), are present in the variable region. A framework region (FR) portion exists between the CDRs to support the CDR ring.

In the present invention, the term "complementarity determining region" is an annular region involved in recognition of an antigen, and the sequence of the region is changed, whereby the specificity of the antibody against the antigen is determined.

As used herein, the term "heavy chain" refers to a full length heavy chain comprising a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3, It means all fragments.

As used herein, the term "light chain" refers to both full length light chains and fragments thereof, including variable region domains VL and constant region domains CL comprising amino acid sequences with sufficient variable region sequences to confer specificity to the antigen.

The antibodies of the present invention were prepared by a fusion cell line (hybridoma) method (see, for example, Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519) by reference to techniques known in the art.

The term "hybrid cell line (hybridoma) " in the present invention means a cell obtained by fusing an antibody-producing B lymphocyte with a myeloma cancer cell. Myeloma cells are transformed cells and can be cultured in a culture dish for a long period of time , Which means that the present invention easily isolates the anti-primate CD19 antibody in large quantities by separating from the myeloma hybrid hybridoma.

In one embodiment of the present invention, the cDNA obtained from the RheCD19 gene of Rhesus monkey is inserted into a retroviral vector (PLNCX2), and then the recombinant vector RheCD19-hIgG / pLNCX2 (SEQ ID NO: 2) expressing the Rhesus monkey CD19 extracellular domain fusion immunoglobulin . Then, the RheCD19-hIgG protein was purified and immunized with the mouse to prepare a fusion cell line. A total of 11 clones were selected for ELISA and a fusion color cell line producing anti-monkey CD19 antibody by dual color FACS analysis. A highly reactive clone was named "RM6H9" and deposited on Jan. 14, 2015 with the deposit number KCLRF-BP-00337 in the Korean Cell Line Research Foundation. The anti - monkey CD19 antibody produced by the fusion cell line was able to specifically detect monkey B cells, cross - react with human CD19, and was found to be effective in analyzing cell subtypes of monkey B cells.

The present invention also provides an anti-primate CD19 antibody produced by the fusion cell line.

The antibody may comprise a variant thereof and the antibody or variant thereof described above may be used in any method that is applicable to the diagnosis of B cell-mediated disease of primate CD19 expressing cells, preferably primate CD19. More specifically, analysis of primate CD19 antigen, quantitative and qualitative analysis of B cells of primate CD19, and the like can be included.

Such modifications include those corresponding to the manufacture of all variants for depletion and inactivation of B cells in vivo in vivo using an anti-primate CD19 antibody. More specifically, it refers to a genetic modification of an antibody and all the modifications resulting therefrom, using a modification of the anti-primate CD19 antibody by chemical conjugation or cDNA sequence information of the antibody.

There are no limitations on the chemical methods and chemical additives for the production of the modified product by the above-mentioned chemical bonding. Specifically, there is no limitation on the use of single chain bacterial toxin (Pseudomonas exotoxin, diphtheria toxin), plant holotoxins (class II ribosome inactivating protein lysine such as ricin, abrin, mistletoe, lectin, moceccin, and hemitoxins (class I ribosome inactivating proteins gelonin, saporin, pokeweed antiviral proteins) That is, the production of a toxin fusion antibody in which a bacterial-derived endotoxin and an exotoxin, which are toxins in the Fc portion of the anti-primate CD19 antibody, are chemically and genetically linked .

Variant production by the above genetic methods includes all forms of single chain and double chain recombinant proteins made using cDNA information and recombinant techniques.

The present invention also provides a method for detecting a primate CD19 expressing cell, comprising contacting an antibody with a sample of a sample to perform an antigen-antibody reaction.

Since the antibody exhibits a higher affinity for the primate CD19 antigen than the conventional antibody, it is excellent for detecting primate CD19 expressing cells, preferably primate CD19 B cells through flow cytometry, fluorescence immunization, immunohistological staining and the like, B cells of CD19 can be isolated with high efficiency. The flow cytometry can analyze various cell characteristics by measuring the size, structure, and granularity of each cell while passing a laser beam through a single flow in a fluid column of a number of cells Method. Very small amount of fluorescent substance can be recognized, and accuracy and sensitivity are high, and a small number of subtypes can be measured or separated in various mixed cell groups. The flow cytometry can be used to detect cell markers such as leukemia marker test using antibodies, growth cycle analysis by measurement of DNA amount of cells, reticulocyte count measurement, neutrophil function test, anti-platelet antibody test and reticular platelet count test, And other tests for research purposes. The use of the antibody of the present invention, which has better affinity with an antigen than an existing antibody, can more accurately and efficiently separate CD19 B cells.

The above-mentioned fluorescence immunoassay refers to a method for determining the quantification and molecular weight of a specific biomolecule and the position where the molecule exists in a cell or a tissue, using the property that the antibody and the antigen selectively bind to each other. In this case, a fluorescent substance is artificially attached to the antibody used, and fluorescence may be generated when light of a specific wavelength is received. An antibody that selectively binds to the molecule can be obtained by using the molecule to be studied as an antigen. It can be used as a means of detecting molecules that have been used as an antigen after binding the obtained antibody to the fluorescent substance so as to be easily explored. If the molecule you are looking for is present in the cell, use a cell slice or special treatment to allow the antibody to pass through the cell membrane, allowing the antibody to react with the molecule. After treatment with antibody, fluorescence of antibody bound to the antigen molecule is observed by fluorescence microscope. As a method for identifying various kinds of molecules constituting a living organism and studying its position and arrangement, it is possible to efficiently select an antibody when an antibody having an excellent binding force with an antigen according to the present invention is used. The sensitivity of the above immunohistochemical staining method is affected by the primary antibody used. The primary antibody can be divided into a polyclonal antibody and a monoclonal antibody. The binding affinity of the primary antibody to the antigen is determined by the ionic interaction, hydrogen bonging, and van der Waals forces between the hypervariable region of the primary antibody and the antigen, Using an antibody of the present invention having an affinity for an antigen more than that of a conventional antibody because an antibody having a high affinity for immunostaining can be expressed as an antibody affinity using a constant (association constant (Ka)), In immunohistochemical staining, primate B19 cells of CD19 can be separated more precisely.

Hereinafter, the present invention will be described in detail by way of examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention in any way to the scope of the invention as defined by the appended claims. It will be obvious.

Example  1. Monkey CD19 - hIg  and Expressed  Production of a fusion cell line overexpressing a fusion cell line and membrane-bound CD19

1-1. Resus  monkey( Rhesus monkey ) Origin CD19  For antibody development CD19 - hIg  Overexpression and Membrane binding type CD19  ( membrane form CD19 ) Construction of over-expressing cell line

In order to construct CD19-hIg overexpression and membrane-bound CD19 (membrane form CD19) over-expressing cell line for the development of Rhesus monkey-derived CD19 antibody, the following experiment was conducted.

More specifically, in order to obtain full length cDNA (1674 bp) and soluble form CD19 cDNA (819 bp) of CD19 of Lethus monkey, peripheral blood mononuclear cells (PBMC) of Lethus monkey were obtained from the project primate center And stimulated with PHA. Thereafter, total RNA was isolated and gene cloning was carried out using gene specific primers. 1 was inserted into a retroviral vector (PLNCX2) using different restriction enzymes, and then the recombinant vector RheCD19 (SEQ ID NO: 2), which expresses the mutant immunoglobulin, -hIgG / pLNCX2 (see Fig. 2). In addition, RheCD19 (whole length) / PLNCX2 also completed the expression vector to obtain membrane-bound CD19 expressing cells (see FIG. 4). And then transformed with CHO cells, which are mammalian cell lines. Briefly, CHO cells were inoculated to a 6-well tissue culture plate at 1 × 10 ⁵ cells per well and cultured in DMEM medium containing 10% calf serum. After 24 hours, if the cells grew to cover 70-80% of the well surface, 1-2 μg of each expression vector DNA was infected with 6-8 μl of LIPOFECTAMINE PLUS (Invitrogen) in serum-free DMEM medium. The transfected 293GPG cells were cultured for 24 hours and the CHO cells were cultured for 4-5 hours using the culture supernatant containing virus (Retro viurs), then cultured for 24 hours in a medium containing 10% serum, Was repeated three times. After that, G418 (1.5 mg / ml) was treated to obtain a stable cell line. Cell lysates were obtained from transformed CHO cells using cell lysis buffer (20 mM Tris pH 7.4, 10 mM EDTA, 0.5% Triton X-100) to confirm that CD19-hIg was overexpressed. After electrophoresis on a 10% polyacrylamide gel, the protein was transferred to Membrane (Amersham Hybond-p, GE Healthcare), the goat anti-human immunoglobulin antibody was bound to the primary antibody and the secondary antibody HRP-conjugated anti-goat immunoglobulin antibody was conjugated with a secondary antibody and developed with Chemiluminescent Substrate (Thermo, 34080) and exposed to a Kodak film (KODAK BioMax Light Film, 8689358). The results are shown in Fig.

As shown in FIG. 3, Western blotting confirmed that the Lysus monkey CD19 fusion immunoglobulin protein of the present invention was expressed in the CHO cells into which the expression vector was introduced.

After confirming the expression of the Rhesus monkey CD19 fusion immunoglobulin protein, the Protein A Agarose kit (553-50-00, manufactured by Takara Shuzo Co., Ltd.) was used to purify the expressed protein from the culture supernatant obtained by culturing the transfected cells. KPL) was used. First, after washing / binding buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4) was passed through Protein A Agarose column, the culture supernatant containing the Rhesus monkey CD19 fusion immunoglobulin protein And adsorbed on the column using the binding force between Protein A and immunoglobulin (IgG). The adsorbed proteins were eluted again with Elution buffer (0.2 M Glycine, pH 3 ± 1.85), purified and used as an antigen for the production of the antibody of Example 2 below.

In order to obtain membrane-bound CD19-expressing cells, the above-mentioned RheCD19 (whole field) / PLNCX2 and the anti-human CD19 antibody (J4.119, Abnova) were incubated at a high concentration (2 ug / test ) And analyzed by flow cytometry. The results are shown in Fig.

As shown in Fig. 5, cell lines in which CO19-CHO cells derived from Rhesus monkey were well expressed were obtained.

1-2. Antigen immunity Immunization )

The CD19-hIg protein isolated in Example 1-1 was immunized under the following conditions.

Immunization term Immune pathway amount Marie Immune Enhancer 2 months - 1 month - 1 month - 3 times in total Abdominal cavity CD19-hlg / 1-5ug / mouse 5 /
Each group ODN

1-3. fusion( fusion )

To produce a fusion cell line, spleen cells obtained from immunized mice were fused with P3X63Ag8.653 mouse myeloma cells and mouse B cells obtained by the antigen immunization of Example 1-2. First, myeloma cells were grown in RPMI medium containing 10% FBS (Roswell Park Memorial Institute medium), washed three times with RPMI medium without FBS (Fetal bovine serum) before cell fusion, Respectively. The spleen cells were harvested after excision of the spleen. The cells were transferred to a tube, allowed to stand for 2 minutes, washed three times, and counted. The myeloma cells and spleen cells were mixed at a ratio of 1: 5, centrifuged and the supernatant was completely removed. 1 ml of 50% PEG-1500 heated to 37 ° C was added slowly over 1 minute, and the tube was gently shaken for 1 minute. Then, 1 ml, 1 ml, and 3 ml of RPMI medium were dropped for 1 minute each, and 14 ml was gradually diluted stepwise while dropping for 2 minutes. After centrifugation at 1200 rpm for 5 minutes, the supernatant was removed, and the pellet of the fused cells was slowly released. Then, RPMI medium (20% FBS, hypoxanthine, aminopterin (aminopterin) and thymine rich in (thymidine)) and, Pipette 200ul in 96-well culture plate, and then, 37 ℃, 5% CO _2, and And cultured in an incubator under the conditions. On day 7 after fusion, the medium was replaced with HT. After 4 ~ 5 days, 150 ~ 200ul of the supernatant was taken from the wells formed in the community and the fusion cell line reacting specifically with monkey PBMC was selected by flow cytometry.

1-4. Of antibody-producing fusion cells Cloning

A supplier cell culture or a medium for a fused cell was prepared using a peritoneal macrophage separated from the abdominal cavity of a mouse, and the fusion cell line was transferred into 24 wells and cultured. When the cultured cell populations grew to about 50 to 70%, the culture supernatant was taken to determine the activity of monkey PBMC. Then, a highly active fusion cell line was diluted in HT medium and cultured twice or three times in a single cell culture with a limiting dilution culture method.

Example  2. ELISA  And dual color FACS  Analysis of anti-monkeys CD19  Selection of fusion cell line producing antibody

The following experiments were conducted to screen for fusion cell lines producing the anti-monkey CD19 antibody through ELISA and dual color FACS analysis.

More specifically, for the sandwich ELISA performed in one step, F (ab ') 2 of clone AE1 (Acris Antibodies, Schillerstraße, Germany) as anti-CD19 antibody was captured using Raji cell lysates as an antigen source capture) and sandwich ELISA. Of the 1056 clones tested, 27 clones were selected as positive. The binding of Raji (B cell) surface CD19 antigen on the surface of the Raji (B cell) cell was reaffirmed by FACS method in order to select antibodies reactive to CD19 in two steps. As a result, as shown in Fig. 6, a total of 11 clones were selected as candidates for the fusion cell line producing the anti-monkey CD19 antibody. Among them, 6H9-4-17 (6H9-4-19) was named RM6H9.

In addition, dual color FACS analysis was performed to screen anti-monkey CD19 antibodies. For this, monkey CD19-CHO stained with a red dye and non-stained CHO cells were stained with an antibody and anti-mouse Ig-APC conjugate was used for antibody detection. The results are shown in Fig.

As shown in FIG. 7, the CD19-specific clones are positive only in the upper right part (CD19-CHO) in the quadrangular dot graph and negative in the lower left (CHO) Two clones (RM6H9 and RM6D9) were selected as CD19 specific clones, and the final monoclone was selected by adding the clone one by one in the same clone.

Through this process, 6H9 (6H9-4-19, 6H9-4-17), a hybridoma cell line (hybridoma) producing the finally selected anti-monkey CD19 antibody, was introduced into Korean Cell Line Research Foundation on Jan. 14, 2015 Deposited under the name RM6H9, and granted accession number KCLRF-BP-00337.

Example  3. Developed monkeys CD19  Usability investigation as a reagent for research and diagnosis of antibodies

3-1. Monkey B cells Flow cell  analysis

To investigate the availability as a research reagent for the selected anti-monkey CD19 antibody, flow cytometry analysis of B cells from PBMCs of Lethus monkeys was performed as follows.

More specifically, as shown in Fig. 8, a 6H9-PE conjugate antibody (A) (0.25 ug / ml) or a commercialized PE-conjugated antibody (B) (J4.119 clone, 0.25 ug / ml) (30 min, ice) with monkey PBMC and analyzed by flow cytometry. At this time, the reactivity to CD4 (C), CD8 (D), and CD16 (E) was also confirmed to confirm that the cells only respond to B cells. The results are shown in Fig.

As shown in FIG. 8, the antibody produced by RM6H9 showed higher affinity for monkey B cells compared to the commercialized antibody (J4.119) when the same amount (0.25 ug / ml) was treated . Further, it was confirmed that the anti-monkey CD19 antibody of the present invention does not detect other CD4 + T, CD8 + T, and CD16 + NK cells. Thus, the anti-monkey CD19 antibody of the present invention can specifically detect monkey B cells Respectively.

3-2. Cross-linking analysis with human B cells

6H9-PE conjugates were made and compared to the commercialized anti-CD19-PE conjugate to see if the antibody produced by the anti-monkey CD19 antibody, RM6H9, reacted with human B cells. Human CEM7 (T cells) and human Daudi (B cells) were mixed and stained with each antibody-conjugate. The results are shown in Fig.

As shown in Fig. 9, it was confirmed that the anti-monkey CD19 antibody according to the present invention cross-linked to human CD19.

3-3. Flow cytometry  Through monkey B cells Cell subtype  Analysis method

PBMCs were isolated from monkey blood to confirm that the RM6H9 antibody of the present invention could actually be used for the subtype analysis of B cells in monkey blood and anti-CD19-PE (6H9), anti-HLADR, anti-CD27, anti-IgM antibody Were stained with ice for 30 minutes, and analyzed by flow cytometry. The results are shown in Fig.

As shown in Fig. 10, primate B cell subtype analysis, native (CD19 + CD27-IgM +), immature (CD19 + CD27-IgM-), mature marginal (CD19 + CD27 + IgD +) and altered isotype switched (CD19 + CD27 + IgD-).

Therefore, it is possible to analyze subtypes of CD19 antibody in blood by combining anti-monkey CD27 antibody and anti-monkey IgM antibody of the present invention with blood, so that analysis of subtypes of B cells and occurrence of B cell-related diseases (Pathogenesis).

Korea Cell Line Research Foundation KCLRFBP00337 20150114

Claims (3)

A fusion cell line (Accession No. KCLRF-BP-00337) that produces an anti-rhesus monkey CD19 antibody recognizing the Rhesus monkey CD19 antigen. An anti-Rhesus monkey CD19 antibody produced by the fusion cell strain of claim 1. A method for detecting a lymphocyte CD19 expressing cell, comprising contacting an antibody of claim 2 with a sample of a specimen to perform an antigen-antibody reaction.

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