KR100295558B1 - Among the primary immune cells, the monoclonal antibody 3-6-A (dendritic cell), which specifically acts on the surface of dendritic cells - Google Patents

Abstract

The present invention relates to the monoclonal antibody 3-6-A which reacts with the extracellular region of the DMa protein.

Description

Among the primary immune cells, the monoclonal antibody 3-6-A, which specifically acts on the surface of dendritic cells

The present invention relates to the monoclonal antibody 3-6-A which reacts with the extracellular region of the DMa protein.

Dendritic cells (DCs) are present in less than 1% of peripheral blood leukocytes (PBLs) in the blood, but when they are exposed to external antigens, such as by pinocytosis or pathogen infection, they absorb the antigen After digesting the protein, the peptide is expressed on the surface of the cell by loading it on MHC class II molecules. Such antigen presentation is much stronger than monocyte or macrophage (Pierre et al ., Nature 388: 787 (1997). When DC is sensitized to external antigens, it secretes DC-specific CC chemokines and returns to the lymph node to activate naive T cells {Adema et al ., Nature 387: 713 (1997); Ingulli E et al ., J. Exp. Med. 185: 2133 (1997)), leading to strong cellular immunity to antigens {Steinman RM Annu. Rev. Immunolo. 9: 271 (1991). In addition, DCs have been reported to be important in the process of positive and negative selection of T cells (Carlos A. Immunol. Today 18), demonstrating that chemokines secreted by DCs act on the mechanism of returning to the lymph nodes of T cells Results have also been reported {Adema et al ., Nature 387: 713 (1997); Ingulli et al., Exp. Med. 185: 2133 (1997).

In addition, it has been shown that cytokine such as interleukin (IL) -12 or CTL that specifically acts on cancer cells is induced to inhibit cancer development and cancer cell proliferation (Gabrilovich et al ., Cell Immunol. 170: 111-9 (1996); Vezzio et al ., Int. Immunol. 8: 19963-70 (1996); Young et al ., J. Exp. Med. 183: 7-11 (1996)). Recently, immunotherapy of cancer using DC has been intensively studied using this property of DC {Gilboa et al., Cancer Immunol. Immunother ., 46: 82-87 (1998); Nestle et al., Nat. Med., 4: 328 (1998); Song et al., J. Exp. Med., 186: 1247 (1997).

It is also known that DCs play a key role in the progression of AIDS (Fauci, Science, 262: 1011 (1993); Pantaleo et al ., Nature, 362: 355 (1993); Embretson et al ., Nature, 362: 359 (1993); Haynes et al ., Science, 271: 324 (1996)}. According to the results of these studies, patients infected with the HIV-1 virus usually undergo asymptomatic infection for 3 to 15 years during which the virus is rarely found in the blood of the patient, Virus and infected CD4 + T cells are found. In some of these reports, DCs are infected by a large amount of virus at the time of primary viremia after HIV-1 infection, and they are returned to the lymph nodes and activate the surrounding inactive T cells, while CD4 + T It is interpreted that the CD4 + T cells are destroyed due to the deficiency of the CD4 + T cells in the blood due to the deficiency of the CD4 + T cells to be supplied to the blood as a result of propagating the virus to the cells, resulting in destruction of the CD4 + T cells {Blauvelt et al ., Clin. Invest. 100: 2043 (1997); McCloskey et al ., J. Immunol . 158: 1014 (1997). Actual, state, etc. {Joo et al ., J. Kor. Soc. Microbiol. , 30: 77 (1995)}, CD4 + T cells are infected with little or no HIV-1, either DC or macrophages, and DCs deliver HIV-1 more efficiently to CD4 + T cells than macrophages Respectively. These results are examples showing that DC plays an important role in the pathogenesis of AIDS.

As the various functions and importance of DC are revealed, much attention has been paid to DC research.

However, since specific surface antigens for DCs have not yet been identified, and various known cell-specific surface antigens are hardly expressed in DCs, it is not easy to purely separate DCs from the blood, However, at present, research on DC is not actively conducted.

Currently the most widely used separation methods are negative selection {Freudenthal et al ., Proc. Natl. Acad. Sci. 87: 7698 (1990). In this method, a complex process such as ficoll gradient, rosetting, metrizamide gradient, adhesive panning, antibody panning, etc., In the leukopak, which collects only leukocytes from blood buffy coat or blood after removing plasma, immune cells such as T cells, B cells, mononuclear cells / macrophages are sequentially removed, and the remaining DC is separated do. However, the process is difficult, complex, and time-consuming to generalize.

In addition to this method, bone marrow stem cells (CD34 +, CD11 +, etc.) or blood precursor cells are isolated and treated with cytokines such as GM-CSF / IL-4 in vitro to differentiate Inducing and differentiating into DC (Bender et al ., J. Immunol. Methods 196: 121-135 (1996). However, this method is costly and very difficult to generalize.

In order to overcome these limitations, it is expected that specific surface markers for DC should be prioritized, and positive selection for DC should be established when producing monoclonal antibodies thereto.

In this regard, several DC-specific surface antigens have been reported so far. However, each of them has limitations, and it is difficult to use DC for positive selection in PBL.

For example, monoclonal antibodies to the HB15 molecule (CD83) react only with activated DCs and not with DCs that are inactive. In addition, although CD83 has been reported to be expressed on activated DCs, it has also been shown to be significantly expressed in activated mononuclear cells / macrophages, being inappropriate as a DC-specific antigen and mainly used for the identification of DCs differentiated by cytokines in vitro (Fearnley et al., Blood, 89: 3708 (1997); Zhou et al., J. Immunol., 154: 3821 (1995)}. In addition, according to the results of experiments conducted by the present inventors, even when activated DCs were expressed, the expression level of CD83 was not sufficient compared with other cell surface antigens.

In addition, CD11c or CD1a has been published as a DC specific antigen {Gao et al ., Immunology 91: 135 (1997); Lardon et al ., Immunology 91: 553 (1997); Ruedl et al ., Immunol. 266: 1801 (1996). However, as well as in DC, CD11c is expressed in macrophages, granulocytes and NK cells, and CD1a is also expressed in thymocytes and Langerhans cells, which is problematic in its specificity. As a result of the experiments conducted by the present inventors, CD11c is expressed in DC but is expressed in a large amount in B cells or mononuclear cells, so that there is a problem in specificity. CD1a is rarely expressed in inactive DC, It was evaluated to be unsuitable for the following.

Thus, the inventors searched the DC cDNA library for DC-specific surface antigens and found that HLA-DMα / b gene (hereinafter, weakly as DM) was specifically expressed in DC of PBL by differential hybridization (Bae et al ., Mol. Cells , 5: 569 (1995)). Based on this, we have succeeded in producing the mAb 3-6-A to the DM protein and confirmed the specificity and reactivity of this mAb 3-6-A to the DC surface antigen Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide monoclonal antibody 3-6-A that specifically recognizes only DCs in PBLs.

Another object of the present invention is to provide a hybridoma cell KHB-DM which produces the aforementioned monoclonal antibody 3-6-A.

FIG. 1 is a reaction flow diagram schematically showing the process of obtaining the monoclonal antibody 3-6-A according to the present invention.

Fig. 2 shows results of SDS-PAGE electrophoresis analysis showing that recombinant DMa is normally expressed in Escherichia coli transformed in Example 3. Fig.

Column 1: Protein size marker,

Column 2: pRSET / A vector only transformed E. coli BL21 (DE3),

Column 3: E. coli BL21 (DE3) expressing recombinant DMa,

Column 4: A sample of column 3 separated pure with Ni ⁺ -NTA resin (QIAGEN).

3 is a photograph showing a positive plaque formed by High-5 cells transfected in a baculovirus system in Example 4. Fig.

4 is a Western blot experiment showing that recombinant DMa is normally expressed in a baculovirus system in Example 8. Fig.

Column M: Protein size marker,

Column 1: High-5 cells not infected,

Column 2: High-5 cells infected with the wild baculovirus Bac-N-Blue,

Column 3: High-5 cells infected with recombinant baculovirus Bac-N-Blue-DMa,

Column 4: E. coli BL21 (DE3) expressing the recombinant DMa of Example 3.

Figure 5 is a Western blot experiment showing the reactivity with normal DMa expressed in Raji cells of mouse antiserum against recombinant DMa in Example 9.

Column 1: As a control, Jurkat cell lysate,

Column 2: Raji cells,

Column 3: Raji cells treated with? -Interferon.

6 is a Western blot experiment showing the reactivity with DMa expressed in cells of monoclonal antibody 3-6-A in Example 12. Fig.

Column 1: As a negative control, Escherichia coli BL21 (DE3), which does not express DMa,

Column 2: Escherichia coli BL21 (DE3) expressing DMa,

Column 3: High-5 cells expressing DMa,

Column 4: Raji cells,

Column 5: DC sample.

FIG. 7 is a fluorescence microscope photograph showing the result of staining PBL cells with the monoclonal antibody 3-6-A in Example 15. FIG.

Panel A: T cells, Panel B: B cells,

Panel C: Mononuclear and macrophages, panel D: DC,

Panel E: Raji cells fixed with organic solvent,

Panel F: Raji cells not immobilized.

8 is a fluorescence microscope image of Example 16, comparing the DC specificity of monoclonal antibody 3-6-A of the present invention with commercialized monoclonal antibodies known to react specifically to DC.

Column 1: T cells, column 2: B cells,

Column 3: Mononuclear cells, Column 4: DC

9 is a dot immunoblot experiment result comparing the DC reaction force of the monoclonal antibody 3-6-A of the present invention with commercialized monoclonal antibodies known to react specifically to DC in Example 17. Fig.

Spot 1: Monoclonal antibody against CD1a

Spot 2: Monoclonal antibody against CD11c

Spot 3: Monoclonal antibody to CD83

Spot 4: Monoclonal antibody against F-actin

Spot 5: Monoclonal antibody 3-6-A

FIG. 10 shows the results of analysis of reactivity between monoclonal antibody 3-6-A and human primary immunocytes by a light-sensitive marker cell separator (FACS) in Example 18. FIG.

Panel A: Cells immediately separated by the method of Example 1,

Panel B: PBLs of Example 1 were cultured for 1 week, and then the cells were purified.

Column 1: T cells, column 2: mononuclear or macrophages,

Column 3: B cells, column 4: DC

The present inventors obtained a monoclonal antibody against the DM protein by expressing the DM protein in E. coli and immunizing the BALB / c mouse in order to obtain a monoclonal antibody against the DM protein that specifically expresses DC in the PBL.

However, none of the monoclonal antibodies obtained recognized normal DMa expressed in DC or Raji cells {Kim et al ., Mol. Cells, 6: 684 (1996).

In contrast, the amino acid sequence of DM was analyzed and it was found that there was more than 75% similarity between human and mouse. This suggests that it is not easy to obtain monoclonal antibodies by immunizing DM with BALB / c mice. In addition, Kim et al . {Kim et al ., Mol. Cells , 6: 684 (1996)) showed that the DM protein was not glycosylated because of the expression of DMa in E. coli, or the protein structure was denatured during the extraction of the recombinant DM protein expressed as an inclusion body, It is judged that the monoclonal antibody does not recognize normal DM? Expressed in DC.

In order to overcome this problem, recombinant DMα protein was obtained using the baculovirus system in the present invention. These recombinant DMα proteins were confirmed to maintain normal DMα antigen expression in Raji cells or DCs, and monoclonal antibody 3 -6-A. ≪ / RTI >

The process of obtaining the monoclonal antibody 3-6-A according to the present invention can be summarized as follows:

(1) preparing a recombinant plasmid pBlueBacHis2A-DMa by cloning a DNA fragment of 609 bp encoding the extracellular region (nucleic acid sequence 122-731) of the DMa gene into the baculovirus system transfer vector pBlueBacHis2A; ;

(2) The recombinant plasmid pBlueBacHis2A-DMα, with virus full-length (full-length) DNA Bac-N-Blue (Invitrogen) into baculovirus, Co in High-5 cells (Invitrogen Corporation) by transfection, the recombinant baculoviruses Preparing the virus Bac-N-Blue-DMa ;

(3) step of the bulk infection with the recombinant baculovirus Bac-N-Blue-DMα, in High-5 cells to obtain a cell extract, a purified recombinant protein DMα the Ni ⁺ -NTA column (QIAGEN Inc.);

(4) Hybrid to produce monoclonal antibody 3-6-A by immunizing a recombinant DMa protein with BALB / c mouse to obtain splenocytes and fusing it with mouse myeloma Sp2 / 0 cells A step of obtaining a donor cell KHB-DM,

(5) Culturing Hybridoma Cells KHB-DM to Produce Monoclonal Antibody 3-6-A

The hybridoma cell KHB-DM, which produced the monoclonal antibody 3-6-A obtained in the above step (4), was deposited at KCTC-0485BP at the GenBank in the Institute of Biotechnology, Korea Advanced Institute of Science and Technology Respectively.

Analysis of isotype and L chain of H chain using monoclonal antibody isotyping kit for monoclonal antibody 3-6-A of the present invention revealed that the H chain was IgG1 and the L chain was κ.

The monoclonal antibody 3-6-A of the present invention showed a strong reactivity to normal DMa expressed in DC and Raji cells, and reacted specifically to only DC among the primary immune cells, and not only the cytoplasm of DC but also the cell surface Strongly stained. This confirms that DMa is specifically expressed only in DC among the primary immune cells, and that it is expressed not only in the cytoplasm but also on the cell surface.

This fact suggests that DM proteins exist only in cells {Karlsson et al., Science, 266: 1569 (1994); Sanderson et al., Science, 266: 1566 (1994). However, existing reports have been searched almost exclusively in Raji cells or other cell lines, and the expression pattern of DM protein in DC has been disclosed for the first time by the present invention. On the other hand, in the experiments of the present inventors, in the case of Raji cells, DM molecules were expressed in large amounts in the cells but not on the cell surface.

The fact that DM molecules are highly expressed on the surface specifically for DC differs from the previous reports that DM molecules act as chaperone proteins in the expression of HLA-class Ⅱ molecules as intracellular components, besides the chaperone function Suggesting different functions and, above all, strongly suggesting that DM proteins can be used as DC surface-specific antigens.

The monoclonal antibody 3-6-A of the present invention can be used to search for DM molecules expressed on the DC surface of PBLs and to study the function of DM molecules in DCs, as well as to select positive DCs among PBL cells have. DC plays an important role in the immune response. In particular, since it is a key cell for the development of immunotherapy for cancer in recent years, development of an easy pure separation method is urgent. Once the positive selection method using the monoclonal antibody 3-6-A according to the present invention has been established, it will be very useful for such studies and will be useful for immunotherapy.

Hereinafter, the step of preparing the monoclonal antibody 3-6-A of the present invention will be described in detail through examples of the present invention, and specificity and reactivity to DCs will be confirmed through various immunoassays.

EXAMPLE 1 Pure Isolation of DC and Other Primary Immune Cells:

Removing the plasma from the volunteers, the blood remaining after blood cells precipitate (Buffy coat) or when supplied with current Nose (leukopak) collecting only the white blood cells in the blood from the Red Cross Blood Center, the existing reporting methods (Bae et al., Mol. Cells, 5, 569, 1995).

Example 2 Extraction of DC mRNA, Synthesis of cDNA fragment for DMa:

MRNA was extracted from the DCs isolated in Example 1 according to the previously reported method (Bae et al ., Mol. Cells , 5, 569, 1995).

The extracted mRNA was mixed with 1 쨉 g of oligo-d (T) primer, and the resultant was mixed with the reverse transcription reaction solution (0.5 R of RNase inhibitor, 4 1 of primary staining reaction buffer, 2 쨉 l of 0.1 mM DTT, 1 쨉 l of 10 mM dNTP) After incubation at 37 ° C for 2 minutes, 5 units of AMV-reverse transcriptase was added and cDNA was synthesized by reacting at 37 ° C for 40 minutes and 45 ° C for 30 minutes.

The synthesized cDNA was used as a template and amplified by PCR using a primer for DMa of the following sequence:

Bam HI-DM alpha sense primer 5'-GAT AA G GAT CC G TCC CTG AAG CTC CTA-3 ' 122-137 Hind III-DM alpha antisense primer 5'-GT TCA AAG CTT TCA CTC CAG CAG ATC TGA GGG-3 ' 732-712

The PCR reaction was carried out at 94 ° C for 1 minute, at 40 ° C for 30 seconds, at 72 ° C for 45 seconds, and then at 94 ° C for 1 minute and 72 ° C for 1 minute, A 609 bp DNA fragment encoding the extracellular region (nucleic acid sequence 122-732) was amplified.

≪ Example 3 > cDNA cloning of DMa:

The DNA fragment amplified by PCR in Example 2 was digested with restriction enzymes Bam HI and Hind III and cloned into pRSET / A (manufactured by Invitrogen) transformed with the same restriction enzymes to prepare a plasmid pRSET-DMα.

The resultant plasmid pRSET-DM? Was transformed into E. coli BL21 (DE3), and then plated on LB (Amp). M-9 broth (Amp) {1% of bactotryptone, Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, 2M glucose, 0.5% thiamine, 1M MgSO ₄ , 1M CaCl ₂ }, IPTG was added at a final concentration of 1 mM at an absorbance of 0.5, and the expression was induced at 37 ° C for 2 hours.

As a result of SDS-PAGE electrophoresis analysis, it was confirmed that the cloned DMα was normally expressed at a molecular weight of 29 kDa in the transformed E. coli (FIG. 2).

Example 4 : Synthesis of recombinant baculovirus expressing DMa:

The DNA fragment amplified by PCR in Example 2 was digested with restriction enzymes Bam HI and Hind III and cloned into pBlueBacHis2A (Invitrogen) which had been transformed by treating with the same restriction enzymes. Then, E. coli BL21 DE3) to produce a recombinant plasmid pBlueBacHis2A-DM? In a positive colony.

The obtained recombinant plasmid pBlueBacHis2A-DMa and baculovirus full-length DNA Bac-N-Blue (Invitrogen), which had been transformed with restriction enzyme Bsu 36 I, were introduced into the cell line using lipofectin (GIBCO / BRL) High-5 cells (Invitrogen) were transfected according to the manufacturer's instructions. After incubation for about 5 to 7 days, the supernatant was collected and re-infected with High-5 cells at a constant dilution concentration. After incubation for 2 days, the cells were covered with 1% low melting point agarose (50 mg / ml X-gal, 2 x TNM-FH medium and 2% low melting point agarose mixed in equal amounts) Lt; / RTI > After culturing, a positive plaque showing blue color was selected (FIG. 3A), and the above procedure was repeated three times to purify the plaque generated by the recombinant virus (FIG. 3B) Cells to obtain recombinant baculovirus Bac-N-Blue-DMa.

Example 5 Confirmation of DMa Cloning of Recombinant Baculovirus:

The recombinant baculovirus Bac-N-Blue-DMa solution collected in Example 4 was collected and the genomic DNA of the recombinant virus was extracted according to the manual of Invitrogen (Bac-N-Blue transfection kit), and the Bac1 / Bac2 primer Bac-N-Blue transfection kit).

Bac1 primer 5'-TTT ACT GTT TTC GTA ACA GTT TTG-3 ' -44 ~ -21 Bac2 primer 5'-CAA CAA CGC ACA GAA TCT AGC-3 ' +794 to +774

In the above experiment, it was confirmed that the recombinant baculovirus contained DMα gene.

Example 6 Expression and purification of DMa of recombinant baculovirus:

The recombinant baculovirus Bac-N-Blue-DMα was infected to High-5 cells to be 5 MOI and incubated for 5 days in a constant temperature humidifier at 27 ° C. The infected cells were harvested and the recombinant DMα protein expressed in the cells was treated with Ni ⁺ -NTA Resin (manufactured by QIAGEN).

Example 7 Immunization of recombinant DMa in BALB / c mice:

The DMα protein purified in Example 6 was mixed with an equal amount of Freund's adjuvant (manufactured by Sigma), and then primary immunized with BALB / c mice at 5 to 6 weeks of age at a concentration of 50 μg per mouse. The mice were immunized twice or three times with 25 μg per mouse per week.

Three days after the third immunization, blood was collected from the eye vein of each mouse to isolate the antiserum.

Example 8 Confirmation of Reactivity of Antiserum with Recombinant DMa:

Proteins prepared by disrupting High-5 cells infected with recombinant baculovirus Bac-N-Blue-DMα were separated by SDS-PAGE, and then transferred to a transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% Of methanol, 1.3 mM of SDS} using a semi-dry gel transfer blotter (Bio-Rad). The membrane was then immersed in a blotto (5% (w / v) skim milk, 0.02% NaN ₃ in PBS), blocked for 30 minutes, washed three times with 1 x PBS, Was allowed to stand in a solution diluted with 10 ml of PBS and allowed to react at room temperature for 30 minutes or longer.

As secondary antibody, anti-mouse-IgG-alkaline phosphatase (Sigma, Bio-Rad, BRL) was used and each step was washed three times with 1 x PBS. As the substrate of the alkaline phosphatase, 66 ㎕ of NBT (Nitro blue tetrazolium: nitroblue tetrazolium) of 50 mg / ml and 5 brom-bromo-4-chloro-3-indolyl phosphate (5-bromo- 4-chloro-3-indolyl phosphate) was mixed with 10 ml of AP buffer (100 mM NaCl, 5 mM MgCl ₂ , 100 mM Tris-Cl, pH 9.5) The reaction was terminated by the addition of a stop solution (20 mM EDTA, 150 mM NaCl in 10 mM Tris-Cl, pH 8.0) when bands appeared on the membrane after reaction in the dark.

The Western blot experiment described above confirmed that the recombinant baculovirus Bac-N-Blue-DMα normally expresses DMα in High-5 cells (column 3 in FIG. 4).

<Example 9> Confirmation of reactivity with DMα expressed in antiserum cells:

Raji cells treated with 100 units / ml of γ-interferon (Sigma) for 3 days were adjusted to a cell number of 1 × 10 ⁶ , respectively. Using the mouse antiserum obtained in Example 7, Western blotting was carried out in the same manner (Fig. 5).

From the above experiments, it was confirmed that the antiserum obtained by immunizing BALB / c mice with recombinant DMa expressed in baculovirus system (Example 7) reacted well with normal DMa expressed in Raji cells.

Example 10 Preparation of hybridoma cells for production of monoclonal antibodies:

Mice immunized with the recombinant DMa expressed in the baculovirus system in Example 7 were euthanized by spinal dislocation, and the spleen was excised and 1.4 x 10 &lt; ⁷ &gt; cells of spleen cells were suspended in 10 ml of high-glucose DMEM medium. Then, the cells were mixed with 3 × 10 ⁶ cells of mouse bone marrow tumor cells: SP2 / 0 cells (Sp2 / 0-Ag14 KTCC CRL1581), washed with a DMEM medium containing high glucose, and 50% PEG-4000 Solution (GIBCO / BRL) was added slowly for 1 minute to induce cell fusion. The DMEM medium of high-content glucose was slowly added at a rate of 1 ml / min to mix the cells well. The supernatant was removed by centrifugation, and the cell pellet was dispersed in 5 ml of 1x DMEM _20% , and the cells _were treated with a feeder cell (Antibody Lab. Manual ed. HD Lane, p220, 1989) was cultured for one day. Then, the same amount of 2 × HAT medium was added, followed by long-term culture, and only fused cells were selected. In this experiment, hybridoma cells that survived in HAT medium and formed colonies in 18 wells were obtained.

Example 11 : Detection of monoclonal antibodies reacting with DMa:

The 18 colonies harvested in Example 10 were each mass-cultured, and the supernatant was analyzed by ELISA, dot immunoblot and Western blotting.

DMα proteins prepared in Examples 3 and 6 were dispensed into each of the 96-well plates to a final concentration of 0.5 μg / ml, allowed to stand overnight at 4 ° C., coated with 0.05% Tween 20 in phosphate buffer Was added and washed twice. (Melting of the of the 0.02% NaN ₃ was added the distilled water), 1% skim latex of the 90㎕ / well of filling which was reacted for 1 hour at After standing for 1 hour at 37 ℃, 37 ℃ the antiserum solution of Example 7. After washing, secondary antibody (chlorine-anti-mouse-IgG-AP: 1/1000 dilution) was added and reacted at 37 ° C for 2 hours. After washing twice, 1 mg of alkaline phosphatase substrate solution { p -nitrophenyl phosphate was added to 10% of diethanolamine buffer (97 ml of diethanolamine, 0.2 g (0.02%) of NaN ₃ , 100 mg of MgCl ₂ .6H ₂ O 0.01% in DDW 800 ml / total 1 L) was added to each well, followed by reaction at 37 캜 for 30 minutes. After completion of the reaction, the absorbance was measured at 405 nm with an ELISA reader. Western blots were performed in the same manner as in Example 8, and dot immunoblots were performed by the method of Kim et al . (Kim et al ., Mol. Cells, 6, 684, 1996).

Table 1 summarizes the above experimental results. In Table 1, the reaction of the monoclonal antibody with the recombinant DMα protein expressed in E. coli and the baculovirus system was examined by ELISA and Western blotting, and the reactivity with other cells was examined by dot immunoblotting and Western blotting .

Binding Ability of Monoclonal Antibody to DMα Antigen antibody Recombinant DMα protein cell E. coli High-5 PBL T cell B cells MC DC Raji DMa pAb ++++ ++++ + - ± - ++ ++ 1-1-H ++ - ± - - - - - 1-3-A ++++ - - - - - - - 1-8-G ++++ - - - - - - - 1-10-A +++ + - - - - - - 1-11-E ++++ - - - - - - - 2-11-G ++++ - - - - - - - 2-12-A ++++ - - - - - - - 3-4-B +++ - - - - - - - 3-5-B ++++ - - - - - - - 3-6-A ++++ +++ + - ± - +++ +++ 4-1-C ++ - - - - - - - 4-2-B ++++ - - - - - - - 5-3-G +++ - - - - - - - 5-5-D ++++ - - - - - - - The binding capacity was measured by ELISA, dot immunoblotting and Western blotting. -: When the result was similar to the negative control, +: ELISA method was used when the standard deviation was 5 times or more When the absorbance at 405 nm is increased by 0.2 as the starting point, the number of codes is added, and when the number of codes is 4 or more, it is represented by "++++". In the case of the Western blot method and the dot immunoblot method, If the signal is clear and repetitive, the number of signs is added as the relative strength increases. ±: The signal is too weak or the result is not repeated.

These results show that all 14 monoclonal antibodies showed a strong reaction with recombinant DMa expressed in Escherichia coli but did not react with DMa expressed in High-5 cells except monoclonal antibody 3-6-A.

Monoclonal antibody 3-6-A reacted strongly with DC in PBL in western blot and dot immunoblot experiments and also showed strong reactivity with Raji cells in Western blot experiments. In Western blot experiments, the B cell fraction sometimes showed a weak positive reaction, presumably because the activated B cells expressed DM in the cells.

These results are summarized as follows. Among the 14 monoclonal antibodies, 3-6-A was selected as a monoclonal antibody against DMα, and the hybridoma cell KHB-DM expressing it was deposited on May 29, 1998, Deposited under accession number KCTC-0485BP.

Example 12 Confirmation of reactivity with mAb expressed in cells of monoclonal antibody 3-6-A:

Using monoclonal antibody 3-6-A, Western blot was performed on recombinant DMa, and DMa normally expressed in Raji cells and DC (Fig. 6).

From the above results, it was confirmed that the mAb 3-6-A reacts well with Raji cells and 34 kDa DMα normally expressed in DCs.

Example 13 Mass Production of Monoliter Antibody 3-6-A:

Hybridoma cells KHB-DM was cultured in DMEM _20% medium containing glucose at a high concentration of 2 × 10 ⁵ cells / ml. After 4 days, when the cells were maximally grown, the supernatant was collected and used as a monoclonal antibody 3-6-A source . At this time, the monoclonal antibody contained in the supernatant is estimated to be about 40 μg per 1 ml of medium.

In order to produce a large amount of monoclonal antibody, 1 ml of pristane was injected into peritoneal cavity of BALB / c mice, and after 1 week, 5 × 10 ⁶ cells of hybridoma cells were injected per mouse. After 10 days, when the abdominal cavity was swollen due to repercussions, the ascites was collected with a syringe (about 10 ml per flask), and then subjected to centrifugation at 1,500 rpm for 10 minutes. To the supernatant, 0.02% NaN ₃ was added and stored in an ultra-low temperature refrigerator at -70 ° C. A monoclonal antibody of about 1 to 9 mg / ml was collected from a mouse.

&Lt; Example 14 > The H chain of monoclonal antibody 3-6-A is IgG1, and the L chain is kappa:

The isotype of the monoclonal antibody 3-6-A produced by the hybridoma cell KHB-DM selected in Example 11 was analyzed using a monoclonal antibody isotyping kit IsoStrip ^TM (manufactured by Boehringer Mannheim).

The isotype of the H chain was IgG1, and the L chain was found to be κ according to the supplier's manual.

Example 15 Monoclonal antibody 3-6-A specifically stained only DCs among PBL cells:

The slide glass for microscope was coated with 1 mg / ml poly-L-lysine (MW 400,000) solution for 15 minutes and washed with distilled water to use as a coating slide.

Raji cells (ATCC CCL86) and T cells, B cells, mononuclear cells, and DCs separated by the method of Example 1 were diluted to 10 ⁵ cells / ml, attached to a coated slide, washed with phosphate buffer, After immersing the cells in an organic solvent (50% methanol + 50% acetone) for about 2 minutes, the cells were washed twice with phosphate buffer, and 100 μl of the monoclonal antibody (culture supernatant) collected in Example 13 was added , And incubated for 1 hour in a CO ₂ incubator. Then, the cells were washed three times with phosphate buffer, and anti-mouse-IgG-FITC was added as a secondary antibody. The cells were incubated at 37 ° C for 30 minutes in a CO ₂ incubator. After washing, the cover glass was covered and observed with a fluorescence microscope (Nikon E-600 Epi-fluorescence microscope).

As shown in FIG. 7, DMα was expressed only in DC among PBLs (FIG. 7D) and was not expressed in T cells, B cells, mononuclear cells and macrophages (FIGS. 7A, B, C), and Raji cells used as a positive control group also had a large amount of DMα expressed therein (FIG. 7E). However, in the case of Raji cells, it was found that the cell surface was not stained with monoclonal antibody 3-6-A at all when the cells were not immobilized with an organic solvent (Fig. 7F). These results are consistent with previous reports that DM molecules are expressed intensely in cells but not on cell surfaces in Raji cells (Karlsson et al., Science 266: 1569-1573, 1994; Sanderson et al., Science 266 : 1566-1569,1994).

Example 16 Identification of Monoclonal Antibody 3-6-A to DC Surface Antigens:

Each of the T cells, B cells, mononuclear cells, and DCs isolated by the method of Example 1 was diluted with phosphate buffer to 10 ⁵ cells / ml, and 100 μl of the monoclonal antibody (culture supernatant) collected in Example 13 was added thereto to prepare 4 Lt; 0 &gt; C for 30 minutes. The supernatant was decanted from 750 g, washed twice with phosphate buffer, suspended in 200 μl of phosphate buffer, and 10 μl (1 μg) of anti-mouse-IgG-FITC (Sigma) as a secondary antibody was added The reaction was carried out at 4 ° C for 30 minutes. The cells were washed twice with phosphate buffer solution and then attached to a coating slide, and observed with a fluorescence microscope used in Example 15.

In FIG. 8, columns 1, 2, 3 and 4 respectively show T cells, B cells, mononuclear cells and DCs reacted with several commercially available monoclonal antibodies and the monoclonal antibody 3-6-A of the present invention, (Anti-CD83-PE) labeled with fluorescence were all stained with secondary antibody labeled with FITC.

8, it can be seen that the monoclonal antibody 3-6-A of the present invention reacts very strongly only on the DC surface, while it hardly reacts with other immune cells, and DMα acts as a primary immune cell , Which is expressed specifically only in DC, and is expressed in large amounts on the cell surface as well as in the cell.

In contrast, antibodies against CD11c or CD83, which are known to specifically react to DCs, respond strongly to B cells or mononuclear cells as well as DCs with low specificity, particularly in the case of CD83, which is known to be expressed only in activated DCs (Zhou LJ , J Immunol., 149, 735, 1992), and the monoclonal antibody (anti-CD83; Immunotech Co.) showed similar affinities to monocytes as well as inactive DCs. F-actin (Yamashiro-Matsumura et al., J. Biol. Chem ., 260, 5087, 1985), which is known to be specifically expressed only in DC, .

From these results, it was confirmed once again that the monoclonal antibody 3-6-A of the present invention has higher specificity for DC and strong affinity than any existing monoclonal antibody.

Example 17 Confirmation of the reactivity of monoclonal antibody 3-6-A against DC:

The reactivity of the monoclonal antibody 3-6-A of the present invention against DC was compared with conventional monoclonal antibodies known to react with DC.

(Kim et al ., Mol. Cells , 6, 684, 1996) with conventional commercialized monoclonal antibodies and the monoclonal antibody 3-6-A of the present invention by pure separation of DCs by the method of Example 1, Respectively.

Monoclonal antibody (supplied from NIH AIDS Research and Reference Reagent Program) to monoclonal antibody (Becton-Dickinson) for CD1a, CD11c, monoclonal antibody against CD83 (Immunotech. Each 1 단 of the monoclonal antibody 3-6-A of the present invention was reacted with 10 ⁵ DCs purely isolated by the method of Example 1 by the method of Example 15.

Cells reacted with the primary antibody were reacted in the same manner as in Example 15 with chlorine-anti-mouse-IgG labeled with alkaline phosphatase (manufactured by Progema), and the cells were transferred to a nitrocellulose membrane using a transcription machine ), And the enzyme reaction was carried out by the method of Example 8. [

As shown in the experimental results of Example 16, the monoclonal antibody 3-6-A showed the strongest reaction with DC (spot 5 in FIG. 9), and spots 1, 2, 3 and 4 in FIG. The monoclonal antibodies to CD1a, CD11c, CD83, and F-actin were reacted with DC. After monoclonal antibody 3-6-A, antibody against CD11c strongly reacted with DC (spot 2 in FIG. 9). However, monoclonal antibodies against CD83 or F-actin did not react with DC in an inactive state.

&Lt; Example 18 >

Primary immune cells isolated by the method of Example 1: T cells, B cells, mononuclear cells and DC were reacted with monoclonal antibody 3-6-A at 4 ° C for 30 minutes. (Sigma) as a secondary antibody and reacted at 4 ° C for 30 minutes. Then, the cells were washed twice with phosphate buffer, and then treated with FACStar Plus (Becton-Dickinson).

Panel A of FIG. 10 is cells directly separated by the method of Example 1, Panel B is PBL of Example 1 and cultured in RPMI _10% medium at about 1 × 10 ⁷ cells / ml for 1 week. , Respectively. Columns 1, 2, 3, and 4 were obtained by staining purely isolated T cells, mononuclear cells, B cells, and DC with a secondary antibody labeled with monoclonal antibody 3-6-A and FITC as described above and analyzed by FACS This is a result.

As shown in columns 1 and 2 of FIG. 10, T cells and mononuclear (macrophage) cells did not react with monoclonal antibody 3-6-A in the case of primary separation and separation after one week of culture.

B cells were not reacted at all in the first separation, but when they were separated after one week of culture, cells that react with the monoclonal antibody 3-6-A were observed (column 3 in FIG. 10).

In the pure DC fraction, more than 60% of the cells reacted strongly with the monoclonal antibody 3-6-A, and there was no significant difference in the responses in the case of the first separation and the separation after one week of culture (FIG. 10 4).

Again, it was confirmed once again that the monoclonal antibody 3-6-A of the present invention specifically reacted with DC.

&Lt; Comparative Example 1 >

DMα expressed in recombinant DMα and Raji cells was examined by Western blotting using each antiserum (anti-DMa-polyclonal antibody) as the primary antibody.

The recombinant DMα expressed in Escherichia coli was well recognized, but the normal DMα protein expressed in Raji cells was not recognized properly. On the other hand, the antiserum obtained by immunization with DMα expressed in baculovirus system reacted well with recombinant DMα as well as normal DMα expressed in Raji cells.

This suggests that recombinant DMa expressed in Escherichia coli has a significant difference in antigenicity from normal DMa, whereas recombinant DMa protein expressed in baculovirus system maintains normal DMa antigenicity expressed in Raji cells or DC Do it. For this reason, Kim et al . (Kim et al ., Mol. Cells, 6, 684, 1996) were found to be unable to produce monoclonal antibodies that normally react with DMα, expressed in Escherichia coli.

The monoclonal antibody 3-6-A of the present invention shows strong reactivity to normal DMa expressed in DC and Raji cells, and reacts specifically with only DC among the primary immune cells, and strongly stains not only the cytoplasm but also the DC surface .

The monoclonal antibody 3-6-A of the present invention can be used to search for DM molecules expressed on the DC surface of PBLs and to study the function of DM molecules in DCs, as well as to select positive DCs among PBL cells have.

Claims (4)

A monoclonal antibody 3-6-A, which specifically acts on the surface of a dendritic cell (DC) in primary immunocytes, produced by hybridoma cells KHB-DM (KCTC-0485BP). Hybridoma cells KHB-DM (KCTC-0485BP) producing monoclonal antibody 3-6-A. The recombinant plasmid pBlueBacHis2A-DM alpha, containing a 609 bp DNA fragment encoding the extracellular region of the DMa gene (nucleic acid sequence 122-732). Recombinant baculovirus Bac-N-Blue-DM? Obtained by co-transfecting the recombinant plasmid pBlueBacHis2A-DM? Of claim 3 and baculovirus full-length DNA Bac-N-Blue into High-5 cells.