WO2007134539A1

WO2007134539A1 - Antibody to autoantigen and/or species high conservation antigen and its preparing method

Info

Publication number: WO2007134539A1
Application number: PCT/CN2007/001652
Authority: WO
Inventors: Jie Tang; Yunbo Wang; Hongzhe Zhou; Minghui Zeng
Original assignee: Institute Of Biophysics Chinese Academy Of Sciences
Priority date: 2006-05-23
Filing date: 2007-05-22
Publication date: 2007-11-29
Also published as: CN1850863A; CN1850863B

Abstract

Antibody to autoantigen and/or antigen highly conserved among species and its preparing method are provided. The method comprises: (1) immunizing NZB/W F1 mouse with a mouse autoantigen and/or heterogeneous antigen having high conservation with mouse autoantigen, and (2) obtaining monoclonal antibody or polyclonal antibody against autoantigen and/or antigen highly conserved among species. The method can be used for manufacturing higher-titer antibody against mouse autoantigen and/or antigen highly conserved between mouse and human.

Description

Antibody against self-antigen and/or highly conserved antigen between species and preparation method thereof

The present invention relates to an antibody directed against a self-antigen and/or a highly conserved antigen between species and a process for the preparation thereof.

Background technique

The use of monoclonal antibodies to treat diseases is currently a hot spot in the pharmaceutical industry, and the development of monoclonal antibody drugs requires an organic cooperation between research and development. In scientific research, it is necessary to confirm the effectiveness of antibody targets, which requires animal testing of monoclonal antibodies against murine antigens. Currently, monoclonal antibodies against mouse proteins are mainly obtained in rats and guinea pigs. However, antibodies to rats and guinea pigs are heterologous proteins in mice, and long-term pharmacodynamic tests produce rejection, while highly conserved proteins in some species are difficult to obtain monoclonal antibodies in rats and guinea pigs. . In terms of development, it is necessary to produce monoclonal antibodies against human proteins, and the ideal targets for some antibody drugs are highly conserved among species, and human-derived proteins are very similar to mouse-derived corresponding proteins. Due to the immune tolerance of mice to their own proteins, highly conserved proteins are difficult to produce a strong immune response when immunized mice as antigens. For proteins with more than 75% homology, it is generally necessary to immunize the corresponding gene knockout mice to obtain high affinity antibodies. However, the construction of knockout mice is difficult. It is difficult to perform corresponding gene knockout for each highly conserved protein that needs to produce antibodies. Some knockout mice cannot survive, or produce immunodeficiency, or can not be used for immunization. . There is therefore a pressing need for a widely applicable method that allows highly conserved antigens between species to produce a better immune response in mice, thereby screening for high affinity antibodies.

Pure New Zealand black mice (New Zealand, Black, NZB) develop autoimmune hemolytic anemia in most 4-6 months after birth. NZB and NZW (New Zealand, white) hybrid F1 progeny animal NZB/W Fl ( NZB X ZW F1 ), B lymphocytes are polyclonal activated, produce excessive antibodies and anti-antigen antigen antibodies, spontaneously similar to lupus Nephritis. Therefore, it is generally considered to be the best natural model for human lupus erythematosus (Aguas, AP, Esaguy, N., Grande, NR, Castro, AP, and Castelo Branco, NA (1999). Accelerat ion of lupus erythematosus- ike processes By low frequency noi se in the hybrid NZB/W mouse model. Aviat. Space Environ. Med. 70, A132-A136 ; Morel, L. and Wakeland, EK (1998) . Suscept ibi lity to lupus nephritis in the NZB/W Model system. Curr. Opin. Immunol. 10, 718-725; Granholm, NA, Graves, K., Izui, S., and Cavallo, T. (1985) . Pathogenic role of anti-DNA antibodies in murine lupus nephritis. J. Clin. Lab Immunol. 113-118).

Invention disclosure

It is an object of the present invention to provide a method of preparing antibodies directed against autoantigens and/or highly conserved antigens between species.

The method for producing an antibody against a highly conserved antigen between autoantigens and/or species provided by the present invention is to immunize NZB/W F1 mice with a mouse autoantigen and/or a heterologous antigen highly conserved with mouse autoantigen. , obtaining polyclonal or monoclonal antibodies directed against autoantigens and/or highly conserved antigens between species.

The mouse autoantigen is a mouse self protein. The heterologous antigen highly conserved with the mouse autoantigen is a heterologous protein having more than 75% amino acid sequence homology to the mouse self protein.

The mouse autologous protein and/or the immunological adjuvant of a heterologous protein having a homologous amino acid sequence of 75% or more with the mouse own protein may be estrogen.

The estrogen may be estradiol, estriol, estradiol or 17 beta estradiol.

In order to increase the antigenicity of the protein of interest, the mouse self-protein and/or the amino- or carboxy-terminus of the heterologous protein having more than 75% amino acid sequence homology with the mouse's own protein is also fused with a Τ cell-specific antigenic determinant. .

The sputum cell-specific antigenic determinant may be part of any heterologous protein that is specifically recognized by mouse sputum cells but is not recognized by mouse sputum cells, such as the sequence of amino acid residues having SEQ ID NO: 1 in the Sequence Listing.

The mouse self-protein may specifically be IL-18; the heterologous protein having 75% or more amino acid sequence homology with the mouse self-protein may specifically be human MIF or human HMGB1.

Also included in the method is the step of purifying a highly conserved antigen multi-cloning antibody or monoclonal antibody against autoantigens and/or species.

Polyclonal antibodies or monoclonal antibodies directed against autoantigens and/or highly conserved antigens between species are also within the scope of the invention.

DRAWINGS

Figure 1A shows the results of the second immunization of NZB/W F1 and wild type mice with recombinant murine IL-18 protein.

Figure 1B shows the anti-immunization of NZB/W F1 mice by second, third and fourth immunization with recombinant murine IL-18 protein. Body weight result

Figure 2A shows the second time of recombinant mouse IL-18 protein in NZB/W F1 mice implanted with estrogen at doses of 0 mg/mouse, 0.62 mg/mouse, 2.5 mg/mouse and 5 mg/mouse. Antibody titer results after immunization

Figure 2B is 0 mg / only, 0.01 mg / only, 0.05 mg / only, 0. 18 mg / only and

Antibody titer results after a third immunization with recombinant mouse IL-18 protein in NZB/W F1 mice implanted with estrogen at a dose of 72 mg/dose

Figure 3 shows the antibody titer results of immunization of NZB/W F1 mice with human MIF protein.

Figure 4 shows the results of antibody titer immunization of NZB/W F1 mice with HMGB1-GST and HMGB1-MT.

The following experimental methods are conventional methods unless otherwise specified. Example 1. Immunization with murine IL-18 protein NZB/W F1 mice obtained higher titer

Three-month-old NZB/W F1 mice (Nanjing University Model Animal Research Institute) and B6 (wild type) mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized as follows: 10 μg recombinant mouse IL- 18 (Biosource International, Inc., Cat. No. PMC0184) Subcutaneous immunization with complete Freund's adjuvant (first immunization), subcutaneous immunization with 5 μg recombinant mouse IL-18 plus incomplete Freund's adjuvant after 14 days and 21 days Secondary (second and third immunizations), after a further 14 days, a fourth immunization was performed twice with 5 micrograms of recombinant mouse IL-18 (once every other day). At the same time, 3 months old NZB/W F1 mice and B6 (wild type) mice were immunized four times with PBS (pH 7.5), and each immunization dose was 0.1 ml/mouse as a control.

Serum was taken 1 day after each immunization, and plated with recombinant mouse IL-18 (10 μg/ml), and antibody titer was measured by ELISA. The results showed that the immune response of NZB/W F1 mice was significantly higher than that of wild-type mice (Fig. 1A), and the serum titer of the fourth immunized NZB/W F1 mice reached 60,000 (Fig. 1B).

1A and 1B, the titer of OD = 0.5 indicates the dilution of the serum when the ELISA reading is 0.5. In Fig. 1A, BW-728, BW-729, BW-730, BW-731 and BW-732 represent antibody titers of five ZB/W F1 mice after the second immunization of PBS; BW-733, BW- 734, BW-735, BW-736 and BW-737 represent antibody titers of five NZB/W F1 mice after the second immunization of recombinant murine IL-18; B6-227, B6-229 and B6-230 indicate Antibody titers of three B6 mice after PBS second immunization; B6-247, B6-248, B6-249 and B6-250 indicate second immunization with PBS Antibody titers of the last four B6 mice. In Fig. 1B, BW733-1st indicates the antibody titer of NZB/W F1 mouse numbered BW-733 after the second immunization of recombinant mouse IL-18; BW733-2nd, BW734-2nd, BW735-2nd and BW736- 2nd indicates the antibody titer of NZB/W F1 mice numbered BW-733, BW-734, BW-735, BW-736 after the third immunization of recombinant mouse IL-18; BW733- IV, BW734- IV BW735-IV and BW736-IV represent the antibody titers of NZB/W F1 mice numbered BW-733, BW-734, BW-735, BW-736 after the fourth immunization of recombinant murine IL-18. Example 2. The sustained release estrogen tablet was implanted subcutaneously into NZB/W F1 mice to increase the immune response of the mice to autoantigens.

A sustained-release estrogen tablet (Innovative Research of America, Sarasota, FL) containing 0.01 to 5 mg of 17 β estradiol was implanted subcutaneously into the mouse neck, each mouse - tablet. After 7 days, immunological and potency assays were performed using the method described in Example 1. The results showed that estrogen could increase the immune response of NZB/W F1 mice to autoantigens, and the dose of estrogen was optimal at 0.72 mg/mouse (Fig. 2A and Fig. 2B).

In Fig. 2A and Fig. 2B, the titer of OD = 0.5 indicates the dilution of the serum when the ELISA reading is 0.5. In Fig. 2A, E-4, E-5, E-6, E-7 and E-8 represent five NZB/W F1 mice implanted with estrogen at a dose of 0 mg/day. Recombinant murine IL-18 Antibody titer results after protein second immunization; E- 9, E- 10, E- 11, E-12 and E-13 indicate five NZB/ implanted estrogen at a dose of 0.72 mg/dose The antibody titer of the W F1 mouse after the second immunization with the recombinant mouse IL-18 protein; E-14, E- 15, E-16, E-17 and E-18 represent 2. 5 mg / only Antibody titer results after a second immunization with recombinant mouse IL-18 protein in five NZB/W F1 mice dosed with estrogen; E- 19, E-20, E-21, E-22 and E- 23 shows the antibody titer results after a second immunization with recombinant mouse IL-18 protein in five NZB/W F1 mice implanted with estrogen at a dose of 5 mg/dose. In Fig. 2B, E3p-666, E3p-667 and E3p-668 represent antibodies after the third immunization of recombinant NZB/W F1 mice by recombinant mouse IL-18 protein at a dose of 0 mg/dose. Efficacy results; E3-670, E3-67K E3-672 and E3-673 represent four NZB/W F1 mice implanted with estrogen at a dose of 0.01 mg/dose, recombinant mouse IL-18 protein, third Antibody titer results after sub-immunization; E3-674, E3-676 and E3-677 represent three NZB/W F1 mice implanted with estrogen at a dose of 0.05 mg/dose. Recombinant murine IL-18 protein Antibody effect after the third immunization Valence results; E3-679, E3-680 and E3-681 represent three NZB/W F1 mice implanted with estrogen at a dose of 0.18 mg/dose after a third immunization with recombinant murine IL-18 protein Antibody titer results; E3-682, E3-683, E3-684, and E3-685 represent four NZB/W F1 mice implanted with estrogen at a dose of 0.72 mg/dose. Recombinant murine IL-18 protein Antibody titer results after the third immunization. Example 3. Immunization of NZB/W F1 mice with MIF protein to obtain high affinity monoclonal antibodies. MIF is an interleukin that is antagonistic to glucocorticoids and is widely involved in the inflammatory response. This protein is a good drug target in diseases such as sepsis, rheumatoid arthritis, and asthma. The MIF amino acid sequence of human and mouse is 89% identical. The mouse produces only a weak immune response to human MIF protein, and MIF knockout mice are required to obtain monoclonal antibodies against MIF. The present invention uses a human MIF protein expressed by E. coli (the coding region of human MIF protein (huMIF) is cloned into the PET-41a vector (Novagen, USA) in which the GST coding region has been deleted, and the cloned plasmid is amplified in DH5α, and then Expressed in BL21. The fusion protein carries 6 Xhis Tag and is purified by M-NTA affinity chromatography.) Immune wild-type Balb/C mice and NZB/W F1 mice, 80 μg MIF plus MPL+TM emulsion Adjuvant ( sigma) foot immunization, once a week, a total of three times. After the third immunization, serum was taken and the GST-tagged human MIF protein (GST-MIF recombinant protein) was used (the coding region of huMIF was cloned into PET-41a vector (Novagen, USA), and the cloned plasmid was expanded in DH5 α. Increase, and then expressed in BL21. The fusion protein with GST Tag, purified by glutathione affinity chromatography.) The plate, GST-MIF recombinant protein concentration of 10 μg / ml, ELISA detection titer. The results showed that the titer in NZB/W F1 mice was much higher than that of the wild type (Fig. 3). In Fig. 3, 1, 2 and 3 represent three NZB/W F1 mice, and 4, 5 and 6 represent three Balb/C mice.

After immunization, NZB/W F1 mice obtained hybridoma fusion with SP2/0 cells in a ratio of 5:1, and 6 hybridomas secreting specific binding to human MIF antibody were obtained after screening. Table 1 shows that the dissociation constants of three of the monoclonal antibodies are at the nM level.

Table 1 Anti-MIF monoclonal antibodies

Clone number 4E10 2A12 10C3

Antibody subtype IgG2a IgG2b IgG2b

Dissociation constant 1 X 10-9M 1 X 10-10M 3 X 10-9M Example 4. Immunization of NZB/W F1 mice with HMGB1 protein with a T cell-specific epitope tag fusion tag resulted in higher titers.

HMGB1 is a nuclear protein that is also secreted outside the cell as an interleukin involved in the inflammatory response. This protein is a good drug target in sepsis. The human and mouse HMGB1 amino acid sequence is 98% identical, and the mouse does not produce an immune response to human leg GB1 protein. The HMGB1 knockout mouse died a few days after birth and is unlikely to be used for an immune response. Therefore, there are currently no monoclonal antibodies against this protein. The present invention expresses fusion protein (HMGB1-MT) of human HMGB1 and Mycobacterium tuberculosis (MT) Psts-1 protein amino acid 326-344 small peptide (DQVHFQPLPPAVVKLSDAL, sequence 1) (human HMGB1 (hu HMGB1) encodes N-terminus The 181 amino acid cDNA and the small peptide coding sequence of SEQ ID NO: 1 were recombined by PCR and cloned into the PET-41a vector (Novagen, USA) in which the GST coding region was deleted. The cloned plasmid was amplified in DH5 α and expressed in BL21. The fusion protein carries 6 X hi s Tag and is purified by Ni-NTA affinity chromatography.) This is used as an antigen to immunize NZB/W F1 mice, which are co-immunized twice: 10 μg HMGB1-MT plus complete Freund's Subcutaneous immunization (first immunization), subcutaneous immunization (second immunization) with 5 micrograms of HMGB1-MT plus incomplete Freund's adjuvant after 14 days. After the second immunization, serum was taken and His6-HMGB1 (the hu HMGB1 encoding N-terminal 181 amino acid cDNA was amplified by PCR, cloned into the PET-41a vector (Novagen, USA) with the GST coding region deleted, cloned The plasmid was amplified in DH5 a and expressed in BL21. The fusion protein was 6 X his Tag and purified by Ni-NTA affinity chromatography. The plate was incubated at a concentration of 10 μg/ml and the titer was determined by ELISA. The results showed that the titer of two immunized mice reached 1: 16000 (Fig. 4). At the same time, GST and human HMGB1 fusion protein (HMGB1-GST) were used. (The cDNA encoding the N-terminal 181 amino acid of hu HMGB1 was amplified by PCR and cloned into PET-41a vector (Novagen, USA). The cloned plasmid was expanded in DH5 ci. Increase, and then express through BL21. The fusion protein carries GST Tag and is purified by glutathione affinity chromatography.) NZB/W F1 mice were immunized according to the above method, and the titer was detected according to the above ELISA method. The plate has a His6-HMGB1 concentration of 10 μg/ml, and the highest titer is 1:4000 (Fig. 4). This result is better than that of wild-type mice, but not enough for screening for hybridomas.

In Fig. 4, the titer 0D=0. 5 indicates the dilution of the serum when the ELISA reading is 0.5; HMGB1-GST-1, HMGB1-GST-2 and HMGB1-GST-3 indicate the NZB of three immunized HMGB1-GST /W F1 mice; HMGB1-MT_1, legs GB1-MT-2 and HMGB1-MT-3 indicate three immunizations NZGB/W Fl mice of HMGB1-GST.

Industrial application

The invention utilizes the characteristics of spontaneous production of autoantibodies in NZB/W Fl mice, avoids the immune tolerance of mice, and makes the mouse autoantigen and the highly conserved antigen between human and mouse can produce better in this mouse. immune response. The method of subcutaneously implanting estrogen in the present invention further breaks the immune tolerance of the mouse and obtains higher immune titer. The present invention produces a recombinant fusion protein with a bacterial antigen peptide targeting murine T cells and a target protein, thereby providing effective T cell help for autoantigen-specific B cells in NZB/W F1 mice. The present invention is widely applicable to the production of antibodies having a relatively high titer against mouse autoantigens and/or highly conserved antigens between humans and mice.

Claims

Claim

A method for preparing an antibody against a highly conserved antigen between autoantigens and/or species, which is to immunize NZB/W F1 mice with a mouse autoantigen and/or a heterologous antigen highly conserved with mouse autoantigen. Polyclonal or monoclonal antibodies directed against autoantigens and/or highly conserved antigens between species are obtained.

2. The method according to claim 1, wherein: said mouse autoantigen is a mouse self-protein; said heterologous antigen highly conserved with mouse autoantigen is more than 75% of mouse self-protein A heterologous protein of amino acid sequence homology.

The method according to claim 2, characterized in that the immunological adjuvant of the mouse self-protein and/or a heterologous protein having more than 75% amino acid sequence homology with the mouse self-protein is estrogen.

4. Method according to claim 3, characterized in that the estrogen is estradiol, estriol, estradiol or 17 beta estradiol.

5. The method according to claim 2, wherein the heterologous protein has 89% or more amino acid sequence homology with the mouse self protein.

6. The method according to claim 2, wherein: the mouse self protein and/or the amino terminus or the carboxy terminus of the heterologous protein having more than 75% amino acid sequence homology with the mouse self protein are further fused. There are sputum cell-specific antigenic determinants.

7. The method according to claim 6, wherein: said sputum cell-specific antigenic determinant is part of any heterologous protein that can be specifically recognized by mouse sputum cells but is not recognized by mouse sputum cells. , or an amino acid residue sequence having SEQ ID NO: 1 in the Sequence Listing.

The method according to any one of claims 2 to 2, wherein: the mouse self-protein is IL-18; and the heterologous protein having more than 75% amino acid sequence homology with the mouse self protein The protein is human MIF or human HMGB1.

9. A method according to any one of claims 2 to 7, characterized in that the method further comprises the step of purifying a highly conserved antigen polyclonal antibody or monoclonal antibody against the autoantigen and/or the interspecies.

10. A polyclonal or monoclonal antibody directed against a self-antigen and/or a highly conserved antigen between species prepared by the method of any one of claims 1-9.