WO2000029442A1 - Verfahren zur herstellung von antikörpern gegen ein polypeptid, von dem nur die kodierende nukleinsäure bekannt ist - Google Patents
Verfahren zur herstellung von antikörpern gegen ein polypeptid, von dem nur die kodierende nukleinsäure bekannt ist Download PDFInfo
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- WO2000029442A1 WO2000029442A1 PCT/EP1999/008678 EP9908678W WO0029442A1 WO 2000029442 A1 WO2000029442 A1 WO 2000029442A1 EP 9908678 W EP9908678 W EP 9908678W WO 0029442 A1 WO0029442 A1 WO 0029442A1
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- polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
Definitions
- proteins can be detected by suitable antibodies.
- antibodies either the proteins can be purified or for example, it is possible to determine the location of the proteins in tissues and cells.
- Antibodies are conventionally produced in such a way that the proteins are first purified from the cells or the tissue or are produced recombinantly with the aid of bacteria or in insect cells or mammalian cells and that these proteins are then used for the immunization of animals. These processes are often very complex and lengthy. In the case of production in bacteria, the proteins produced in this way are often not identical to the naturally occurring proteins, since the secondary structure can differ from the native proteins and because bacteria do not have the same post-translational modification mechanisms that are present in eukaryotic organisms.
- the present invention therefore relates to a method for producing antibodies which react specifically with a polypeptide from which the coding nucleic acid is known, in which
- the DNA coding for the polypeptide is expressed in a host cell with the aid of a vector which has at least one sequence coding for a detection signal, and the expressed polypeptide is bound to a solid phase with the aid of the detection signal,
- step b) regardless of step a) the DNA coding for the polypeptide is introduced directly into an animal, whereby a Expression of the polypeptide takes place in the animal, which causes the formation of antibodies against the polypeptide and
- step b) the antibodies formed in step b) are reacted with the polypeptide formed in step a) and detected or enriched.
- the method according to the invention essentially consists of three steps.
- the DNA coding for the polypeptide is expressed with the aid of a vector in a suitable host cell (step a)). Since the polypeptide expressed with the aid of the vector is generally only present in a relatively low concentration in the host cell, the vector used according to the invention is provided with a nucleotide sequence which codes for a discovery sequence (tag sequence). This tag sequence is linked to the sequence coding for the polypeptide, which means that the expressed polypeptide has this discovery peptide sequence, for example at the C-terminus.
- step b) which is carried out independently of step a), the DNA coding for the polypeptide is introduced into a suitable animal and brought to expression there.
- the genetic immunization used according to the invention enables the direct formation of antibodies in a host animal.
- purified DNA which contains the genetic information for the protein to be examined and suitable control elements, is injected directly into the organism intended for antibody production (mouse, rabbit, etc.).
- the DNA is taken up by cells of the recipient organism and the protein is expressed in native form (ie with correct post-translational modifications).
- the protein foreign to the recipient organism causes the immune system to produce antibodies directed against the foreign antigen (humoral Immune response).
- This method has already been used successfully for the production of high-affinity, monoclonal antibodies which recognize native proteins
- the expression vectors used for the genetic immunization in step b) for the production of the desired antibodies should also be used in vitro for the production of the target protein.
- the expression vectors are introduced into suitable target cells, in particular mammalian cells, which then synthesize the desired protein. These cells (intact or after lysis with suitable buffers) or media supernatants (in the case of secreted proteins) are intended to serve to detect the protein-recognizing antibody by FACScan analyzes (in the case of cellular proteins) or ELISA.
- the expressed polypeptide when a foreign polypeptide is expressed in a host cell, the expressed polypeptide can be secreted using a secretion or leader sequence. In these cases it is important that the expressed and secreted polypeptide have a detection signal so that the polypeptide can be isolated from the medium. However, if the polypeptide is not channeled outside but remains on the surface of the cell membrane, an additional detection sequence is not absolutely necessary. In this case, the site of the polypeptide that is responsible for the anchorage between the polypeptide and the cell takes over the function of the discovery sequence. In this case, since the expressed polypeptide remains connected to the cell, the antibodies formed can be detected by binding to the polypeptide and subsequent reaction with a fluorescence-labeled antibody by FACScan analyzes.
- a cell ELISA is also possible, in which the bound antibodies are coupled to an enzyme Secondary antibodies and a suitable substrate reaction can be detected.
- the anchoring sequence is a signal sequence which is responsible for membrane anchoring by means of a glycosylphosphatidylinositol (GPI) residue
- the corresponding expression plasmid can be used both for DNA immunization and for detection of the antigen-specific antibodies which have arisen, for example after transient transfection .
- GPI anchor is that it is easily cleaved enzymatically from the cell surface in vivo and thus, as is known for secreted proteins, a good antibody response can be achieved (see Example 7 for a good immune response after genetic immunization with an expression plasmid, which codes for a GPI-anchored protein).
- Sequence suitable complexed Ni 2+ ions to fish out the protein from the cell supernatant or cell lysate.
- Short and / or less immunogenic peptide sequences are particularly suitable as the tag sequence.
- Mouse proteins which stimulate the. Can also serve as less immunogenic tag sequences (for the production of antibodies in mice)
- Antibody production work e.g. GM-CSF, IL-4, IL-10 etc.
- tags have the advantage of not developing an immune response due to the tolerance of the immunized animal to these self-proteins. If the formation of the antibodies recognizing the tag sequence of the recombinant protein cannot be prevented, these can be identified with the aid of constructs which code for irrelevant proteins provided with an identical tag.
- the immobilized protein produced by transient transfection now serves to bind the antibodies which recognize it from the serum or hybridoma culture supernatant (in the production of monoclonal antibodies).
- the detection of the bound specific antibodies is then carried out via enzyme-linked anti-antibodies (detection antibodies), which can be quantified using a specific substrate conversion, usually photometrically.
- detection antibodies enzyme-linked anti-antibodies
- the specificity and sensitivity of the detection system can be significantly increased when using peptide tags if F (ab) 2 fragments of the anti-tag antibody are used as the capture antibody and an Fc region-recognizing antibody is used as the detection antibody.
- This configuration of the ELISA prevents cross detection of the catcher antibody.
- the transcription unit coding for the polypeptide can have a polyadenylation sequence at the 3 'end which is necessary for the stabilization of a eukaryotic mRNA.
- the vector In order for the polypeptide to be expressed in the host cell, the vector usually has a promoter, with strong promoters preferably being used.
- the promoter of the elongation factor l ⁇ or the promoter of the cytomegalovirus can be mentioned as examples.
- the nucleic acid coding for the polypeptide is introduced directly into an animal in order to generate antibodies against the polypeptide there.
- the DNA used for this is in the form of a vector which is selected such that it can be used simultaneously for the two steps a) and b).
- the DNA coding for the polypeptide is introduced by using a so-called gene gun.
- the gene gun microscopic gold particles with the DNA, preferably the vector or Plasmid DNA encased and shot on the shaved skin of the animal. The gold particles penetrate the skin and the DNA applied to them is expressed in the host animal.
- Laboratory animals such as mice, rats or rabbits are preferably used according to the invention.
- genetic adjuvants are also applied in a preferred embodiment together with the DNA coding for the polypeptide.
- plasmids expressing cytokines such as GM-CSF, IL-4, IL-10) that stimulate the humoral immune response in the laboratory animals.
- hybridoma cells are particularly useful when the laboratory animal used is a mouse or rat.
- the immunized mice are sacrificed, spleen cells are isolated and fused with tumor cells and then those clones are selected which secrete the desired monoclonal antibodies.
- the polypeptides to be examined are secreted by the host cells in step a). Since a detection signal is associated with the polypeptides, the polypeptides sought can be isolated by forming a bond between the detection signal (tag sequence) and a suitable ligand.
- the tag sequence is preferably bound to a solid phase. These can be the walls of microtiter plates, gel beads or magnetic beads (so-called magnetic beads).
- the magnetic beads have the advantage that the solution containing the expressed polypeptide can be easily mixed with the magnetic beads.
- the magnetic beads have a ligand (for example antibody fragments) that binds to the tag sequence.
- the magnetic beads can then be enriched by applying a magnetic field. By choosing suitable conditions the polypeptide sought can then be eluted again from the magnetic beads if the antibodies are to be enriched.
- the present application also relates to those antibodies which can be obtained by the process according to the invention.
- FIG. 1 shows the detection of anti-hp70 antibodies in the serum and in the culture supernatant of hybridomas obtained from mice immunized from lymph nodes hp70-pcDNA3-DNA with the aid of FACScan analysis.
- FACScan analysis either untransfected (gray curves) or transiently transfected with hp70-pcDNA3-DNA BOSC cells (white curves) were used.
- GV114 mouse immunized with the hp70-pcDNA3 expression vector. The experiment is explained in more detail in Example 7.
- Promoters eg the promoter of the elongation factor 1 ⁇ [EF-l ⁇ ] gene
- the cDNA region coding for the extracellular domain of thyroid peroxidase (TPO) in humans (2602 bp; 859 amino acids) was cloned into the BamH1 / EcoRV cleavage sites of the polylinker sequence and at the 3 ' end also provided with a region coding for a His ⁇ -ag and a subsequent stop codon (TPO sol. -His-pcDNA3).
- the plasmid DNA was grown in E. coli and purified using a Qiagen plasmid isolation kit (Qiagen, Hilden).
- Expression plasmid-encoded protein can be produced.
- the expression construct was brought into BOSC23 cells by transfection (Pear et al. F (1993) PNAS, 84, 8392-8396].
- the BOSC23 cells is a modified adenovirus 5 transformed human embryonic Renal cell line (HEK293), which is very transiently transfectable. The cells were plated in 6-well cell culture dishes so that they reached 80% confluence the next day.
- DMEM Dulbecco 's modified Eagle 's medium
- FCS fetal calf serum
- the medium was replaced by 5 ml DMEM / 5% FCS. After a further 48 h (72 h after transfection), the cell culture supernatant was removed and stored at -70 ° C.
- TPO sol.-His His 6 -day protein produced by transient transfection to nickel chelate microtiter plates (DÜNN, Asbach)
- the wells were each provided with 200 ⁇ l of supernatant from the transient transfection batch (see above) or one; n ⁇ c. fc-transfected BOSC23 culture supernatant incubated overnight at 4 ° C., then four times with buffer A (50 mM Tris / HCl pH 7.5, IM NaCl) and twice with buffer B (phosphate buffered saline (PBS), 0.1% BSA, 0.05% Tween 20).
- buffer A 50 mM Tris / HCl pH 7.5, IM NaCl
- buffer B phosphate buffered saline
- BSA phosphate buffered saline
- Non-specific binding sites were then blocked by incubation with 300 ⁇ l of 3% bovine serum albumin (BSA) / PBS for 1 h at room temperature and the washings were repeated with buffers A and B.
- BSA bovine serum albumin
- the preimmune and immune sera of the immunized mice were diluted 1: 100 with Buffer B.
- 100 ⁇ l of the diluted mouse sera were placed in the wells of the Given nickel chelate microtiter plates. After a one hour incubation at room temperature, the wells were washed four times with buffer C (50 mM Tris / HCl pH 7.5, 0.5 M NaCl, 0.1% BSA, 0.05% Tween 20), twice with buffer B.
- the specific antibodies directed against TPO were "classically" detected using a commercially available TPO antibody ELISA (Varelisa TPO Antibodies; Pharmacia-Upjohn, Freiburg).
- Anti-TPO antibody is detected in this test system by purified recombinant TPO.
- the anti-TPO antibody content of the preimmune and immune sera of the immunized mice was determined in a dilution of 1: 100 according to the manufacturer's instructions.
- Table 1 Detection of anti-TPO antibodies in the serum TPO sol.-His-pcDNA3-DNA of immunized mice with the help of purified TPO protein (Varelisa TPO Antibodies detection system). Mouse optical density 450 nm
- the preimmune and immune serum of a mouse (GV1 from Table 1) was examined as an example.
- Table 2 at a serum dilution of 1: 100 anti-TPO antibodies can be clearly detected in the immune serum, while the pre-immune serum showed no reaction.
- Table 2 Detection of anti-TPO antibodies in the serum of a mouse immunized with TPO sol. His pcDNA3 DNA using TPO sol. His protein generated by transient expression.
- the ubiquitously active promoter of the elongation factor 1 ⁇ (EF-l ⁇ ) gene was used for expression control.
- the expression vector used is based on the pBluescript vector (Stratagene, Heidelberg), in which a 1.2 kb fragment of the human EF-l ⁇ gene promoter, a 0.7 kb EcoRI fragment with the polyadenylation signal of the human G-CSF cDNA (Mizushima and Nagata, 1990), and between the BaiwHI / iV ⁇ tl interfaces which are responsible for the influenza virus hemagglutinin (HA) - tag-coding oligonucleotide sequence were inserted.
- HA hemagglutinin
- the human cDNA region (431 bp; 135 amino acids) coding for the extracellular domain of the activin receptor IIA (431 bp; 135 amino acids) was cloned into the Clal / BamHI cleavage sites of the polylinker sequence in such a way that the HA-coding region and a subsequent region were found at the 3 ' end Stop codon came to rest (pEF-l ⁇ -ActRII-HA).
- the protein encoded by the expression plasmid pEF-l ⁇ -ActRII-HA was produced by transient transfection of BOSC23 cells as described in Example 2.
- the wells were first coated with the F (ab) 2 fragment of the anti-HA-ag antibody.
- 150 ⁇ l of the antibody fragment were added to each well of the microtiter plate and washed at room temperature with PBS and free protein binding sites were blocked by incubation with 200 ⁇ l of 0.2% BSA / PBS.
- the supernatant of the transient transfection batch (see Example 5) or a mock-transfected BOSC23 culture supernatant was then incubated for 2 h at room temperature, then washed three times with phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- the preimmune and immune sera of the immunized rabbits were diluted 1: 100 and 1: 500 with 0.2% BSA / PBS. 100 ⁇ l of the diluted rabbit sera were added to the wells of the coated microtiter plates.
- hp70-pcDNA3 pcDNA3
- the human hp70 amino acid sequence corresponds to the murine hp70 sequence in approximately 70% of the residues.
- mice with the gene gun were carried out according to a short protocol (6 immunizations within 13 days) as described by Kilpatrick et al. (1998), Hybridoma 17: 569-576.
- lymphocytes from the regional (axillary, brachial, inguinal and popliteal) lymph nodes of three mice were isolated and fused according to a standard protocol with exponentially growing SP2 / 0 mouse myeloma cells (American Tissue Type Culture Collection) with the aid of polyethylene glycol (Sigma) (Campbell AM (1986).
- Monoclonal antibody technology The production and characterization of rodent and human monoclonal antibodies. Book series: Laboratory Techniques in Biochemistry and Molecular Biology (RH Burdon and PH van Knippenberg, eds.), Elsevier Science Publishers, Amsterdam) .
- Lymph node lymphocytes were plated per well of a 96-well microtiter plate and cultured in 100 ⁇ l hypoxanthine / aminopterin thymidine (HAT) -containing DMEM medium (Sigma) with 20% FCS and 5% hybridoma enhancing factor (Sigma).
- HAT hypoxanthine / aminopterin thymidine
- Candidate hybridoma clones were identified using a cell ELISA.
- BOSC cells as described in Example 2, were transiently transfected with the hp70-pcDNA3 expression construct, resuspended in 4% formaldehyde in PBS and fixed for 10 min. The cells were then diluted 1:10 with PBS and stored at 4 ° C. for up to four weeks.
- 96-well round-bottom microtiter plates were blocked by adding 300 ⁇ l of 1% BSA in PBS for 1 h at room temperature. After the solution had been removed by inversion of the plate, 75 ⁇ l of the hybrid o cell supernatant and 10 ⁇ l of transiently transfected BOSC cell suspension (6 ⁇ 10 6 cells / ml of 1% BSA in PBS) were added and incubated at 4 ° C. for 1 h. After adding 100 ⁇ l of 1% BSA in PBS, the mixture was centrifuged at 300 ⁇ g for 4 min and the supernatant was dumped as above.
- the cells were washed again with 200 ⁇ l of 1% BSA / PBS, resuspended in 75 ⁇ l, peroxidase-coupled goat anti-mouse immunoglobulin antibody (DAKO), l: 2,000 diluted in 1% BSA / PBS, and left for 1 h incubated at 4 ° C. Then 100 ul 0.1% Tween 20 / PBS were added and centrifuged as above and the supernatant discarded. The cells were then washed three times with 200 ul 0.1% Tween 20 / PBS and twice with 200 ul PBS.
- DAKO peroxidase-coupled goat anti-mouse immunoglobulin antibody
- Table 3 Detection of anti-hp70 antibodies in the serum and in the culture supernatant of hybridomas obtained from mice immunized from lymph nodes hp70-pcDNA3-DNA using a cell ELISA. BOSC cells transiently transfected with hp70-pcDNA3-DNA were used for the cell ELISA.
- Preimmune serum GV114 1 100 0.08
- FIG. IB shows the histograms obtained for an irrelevant negative control used as negative control (26/3/13) and for the positive hybridoma supernatant N1F4 with BOSC cells transiently transfected or not transfected with the hp70-pcDNA3 expression vector.
- FIG. 1A the histograms obtained in the same test for the immune and preimmune serum of a mouse used for hybridoma production are shown (FIG. 1A). All 20 selected hybridoma supernatants were found to be positive in the FACScan analysis.
- the immunoglobulin class of the hp70-specific antibodies was determined in 19 of the 20 supernatants. Two of the supernatants tested contained hp70-specific IgM antibodies, 17 supernatants contained hp70-specific IgG antibodies.
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000582427A JP2002530065A (ja) | 1998-11-16 | 1999-11-11 | コードする核酸が公知であるポリペプチドに対する抗体を産生させる方法 |
AU11621/00A AU768631B2 (en) | 1998-11-16 | 1999-11-11 | Method for producing antibodies acting against a polypeptide that only recognises the coding nucleic acid |
NZ511040A NZ511040A (en) | 1998-11-16 | 1999-11-11 | Method for producing antibodies acting against a polypeptide that only recognises the coding nucleic acid |
EP99972225A EP1131355A1 (de) | 1998-11-16 | 1999-11-11 | Verfahren zur herstellung von antikörpern gegen ein polypeptid, von dem nur die kodierende nukleinsäure bekannt ist |
CA002350078A CA2350078A1 (en) | 1998-11-16 | 1999-11-11 | Method for producing antibodies acting against a polypeptide that only recognises the coding nucleic acid |
IL14246599A IL142465A0 (en) | 1998-11-16 | 1999-11-11 | Method for producing antibodies acting against a polypeptide that only recognizes the coding nucleic acid |
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DE19852800.0 | 1998-11-16 | ||
DE19852800A DE19852800C1 (de) | 1998-11-16 | 1998-11-16 | Verfahren zur Herstellung von Antikörpern gegen ein Polypeptid, von dem die kodierende Nukleinsäure bekannt ist |
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WO2000029442A1 true WO2000029442A1 (de) | 2000-05-25 |
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PCT/EP1999/008678 WO2000029442A1 (de) | 1998-11-16 | 1999-11-11 | Verfahren zur herstellung von antikörpern gegen ein polypeptid, von dem nur die kodierende nukleinsäure bekannt ist |
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EP (1) | EP1131355A1 (de) |
JP (1) | JP2002530065A (de) |
AU (1) | AU768631B2 (de) |
CA (1) | CA2350078A1 (de) |
DE (1) | DE19852800C1 (de) |
IL (1) | IL142465A0 (de) |
NZ (1) | NZ511040A (de) |
WO (1) | WO2000029442A1 (de) |
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JP2003043044A (ja) * | 2001-07-30 | 2003-02-13 | Denka Seiken Co Ltd | 抗体捕捉法による抗体の免疫学的測定方法 |
US6800462B2 (en) | 2001-09-10 | 2004-10-05 | Abgenomics Corporation | Production of recombinant proteins in vivo and use for generating antibodies |
WO2006016999A1 (en) | 2004-07-09 | 2006-02-16 | Cythera, Inc. | Methods for identifying factors for differentiating definitive endoderm |
WO2007066823A1 (ja) * | 2005-12-07 | 2007-06-14 | Nosan Corporation | 結合組織増殖因子に対する抗体又はそれを含む組成物 |
WO2008044754A1 (fr) | 2006-10-06 | 2008-04-17 | Takeda Pharmaceutical Company Limited | Agent prophylactique ou thérapeutique pour le cancer |
US7510876B2 (en) | 2003-12-23 | 2009-03-31 | Cythera, Inc. | Definitive endoderm |
US7541185B2 (en) | 2003-12-23 | 2009-06-02 | Cythera, Inc. | Methods for identifying factors for differentiating definitive endoderm |
EP2070546A1 (de) | 2004-04-09 | 2009-06-17 | Takeda Pharmaceutical Company Limited | Vorbeugungsmaßnahmen/Heilmittel für Krebs |
US7625753B2 (en) | 2003-12-23 | 2009-12-01 | Cythera, Inc. | Expansion of definitive endoderm cells |
US7695963B2 (en) | 2007-09-24 | 2010-04-13 | Cythera, Inc. | Methods for increasing definitive endoderm production |
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WO2010096941A1 (en) * | 2009-02-24 | 2010-09-02 | Esbatech, An Alcon Biomedical Research Unit Llc | Methods for identifying immunobinders of cell-surface antigens |
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DE10314412A1 (de) * | 2003-03-28 | 2004-10-14 | Genovac Ag | Genetische Immunisierung mit multiplen Expressionskonstrukten zur Herstellung von monoklonalen Antikörpern |
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- 1999-11-11 IL IL14246599A patent/IL142465A0/xx unknown
- 1999-11-11 CA CA002350078A patent/CA2350078A1/en not_active Abandoned
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IL142465A0 (en) | 2002-03-10 |
CA2350078A1 (en) | 2000-05-25 |
JP2002530065A (ja) | 2002-09-17 |
AU1162100A (en) | 2000-06-05 |
EP1131355A1 (de) | 2001-09-12 |
AU768631B2 (en) | 2003-12-18 |
NZ511040A (en) | 2003-07-25 |
DE19852800C1 (de) | 2000-04-13 |
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