THIERAUCH KARL-HEINZ (DE)
MARTINY-BARON GEORG (DE)
TOTZKE FRANK (DE)
MENRAD ANDREAS (DE)
THIERAUCH KARL HEINZ (DE)
MARTINY BARON GEORG (DE)
TOTZKE FRANK (DE)
| WO1995021868A1 | 1995-08-17 | |||
| WO1994011499A1 | 1994-05-26 |
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B. TERMAN ET AL.: "Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 187, no. 3, 30 September 1992 (1992-09-30), DULUTH, MN, USA, pages 1579 - 1586, XP002052457
J. WALTENBERGER ET AL.: "Different signal transduction properties of KDR and flt1, two receptors for vascular endothelial growth factor.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 43, 28 October 1994 (1994-10-28), BALTIMORE, MD, USA, pages 26988 - 26995, XP002052458
B. MILLAUER ET AL.: "High affinity VEGF binding and developmental expression suggest flk-1 as a major regulator of vasculogenesis and angiogenesis.", CELL, vol. 72, 26 March 1993 (1993-03-26), CAMBRIDGE, MA, USA, pages 835 - 846, XP002052459
A. MENRAD ET AL.: "Novel antibodies directed against the extracellular domain of the human VEGF-receptor type II.", HYBRIDOMA, vol. 16, no. 5, 1997, NEW YORK, NY, USA, pages 465 - 471, XP002052460
| 1. | Monoclonal antibodies that are directed against an epitope located within extracellular domains 1 7 of human VEGFreceptor KDR. |
| 2. | Monoclonal antibodies according to claim 1 , characterised in that the anti bodies are directed against an epitope located within extracellular domains 6 and 7 of human VEGFreceptor KDR. |
| 3. | Monoclonal antibodies according to claim 2, characterised in that they are the antibodies AM 279, AM 2101 , AM 512, AM 51013 and AM 241. |
| 4. | Monoclonal antibodies according to claims 2 and 3, characterised in that they are the antibodies AM 279 and AM 2101. |
| 5. | Use of the monoclonal antibodies according to claims 1 to 4 in Western blots, immunoprecipitation, ELISA, FACS analysis and in indirect immunofluorescence microscopy. |
| 6. | Use of the monoclonal antibodies according to claims 1 to 4 in immuno histochemistry. |
| 7. | Use of the monoclonal antibodies according to claims 1 to 4 in screening for small agonistic and antagonistic molecules and in the detection of mutant receptor subtypes. |
| 8. | Use of the monoclonal antibodies according to claims 1 to 4 in diagnostics. |
| 9. | Use according to claim 8, characterised in that the monoclonal antibodies are coupled in combination with a suitable contrastenhancing substance and are used in that form. |
| 10. | Use according to claims 8 and 9, characterised in that the contrastenhancing substance coupled to the antibody is 99m technetium. |
| 11. | Use of the monoclonal antibodies according to any one of claims 1 to 4 coupled to suitable toxic substances. Use according to claim 11 in angiogenesisdependent phenotypes, such as tumours, rheumatoid arthritis or psoriasis Use according to claims 11 and 12 in the case of metastases Use of mRNA that codes for the heavy and the light chain of the antibodies according to the invention in the preparation of recombinant "singlechain antibodies". Use of the recombinant antibodies encoded by mRNA according to claim 14 in Western blots, immunoprecipitation, ELISA, FACS analysis, in indirect immunofluorescence microscopy and in immunohistochemistry A method of determining human VEGFreceptor KDR in cell lysates or tissue analysates, characterised in that 1 the monoclonal antibodies AM 279, 2101 , AM 512, AM 51013 or AM 241 , as captor antibodies, are coupled in purified form at a concentration of 1 10 μg/ml in coupling buffer on ELISA plates and then excess binding sites are blocked with blocking buffer, 2 cell or tissue analysates are prepared in a suitable lysis buffer, 3 the plates are washed in washing buffer before application of the lysates, 4 the KDRprotein to be determined in the sample is quantified using a recombinant KDRprotein as standard curve, 5 the tissue and cell lysates to be analysed are then introduced into the test system, 6 incubation is carried out at room temperature for 2 hours, 7 the ELISA plates are then washed, 8 determination of the "captured" KDR is carried out by means of a polyclonal antiKDR antiserum and finally 9 detection by means of a chromogenic, chemiluminescent or radioactive substance is carried out. |
| 12. | 17 Method according to claim 16, characterised in that the method is carried out in the form of a kinase test procedure that determines tyrosine phosphorylated KDR. |
| 13. | 18 Method according to claims 16 and 17, characterised in that recombinant phosphorylated KDR is used as the standard protein. |
| 14. | 19 Method according to claims 16 to 18, characterised in that the polyclonal anti KDR antiserum used in step 8 of the method is polyclonal anti phosphotyrosine antiserum or a monoclonal antiphosphotyrosine antibody. |
| 15. | 20 Method according to claims 16 to 19, characterised in that the substance used for detection in step 9 of the method is a peroxidaselabelled secondary antibody with suitable chromogenic or chemiluminescent substrates. |
| 16. | 21 Method according to claims 16 to 20, characterised in that the lysis buffer used in step 2 of the method contains from 1 to 5 mM divalent ions and from 1 to 15 % glycerol. |
| 17. | 22 Method according to claims 16 to 20, characterised in that the lysis buffer used in step 2 of the method contains from 1 to 2 mM divalent ions and from 5 to 12 % glycerol. |
| 18. | 23 Method according to claims 16 to 20, characterised in that the lysis buffer used in step 2 of the method contains 1.5 mM divalent ions and 10 % glycerol. |
| 19. | Method according to claims 21 to 23, characterised in that the divalent ion is magnesium. |
The present invention relates to monoclonal antibodies, secreted by hybridoma cell lines, that are directed against an epitope of the extracellular domain of human VEGF-receptor KDR (tønase insert domain containing receptor), to methods of deter¬ mining human VEGF-receptor KDR in cell lysates or tissue analysates and to the use of the antibodies in analytical assays, in diagnostics and as carrier molecules for therapeutic substances.
New vessels form as capillaries which sprout from existing small vessels. This process, known as angiogenesis, takes place in response to certain signals (Alberts et al., Molekularbiologie der Zelle, Verlag Chemie, Weinheim).
Angiogenesis is a process that is meticulously controlled by the interaction of vascular endothelial growth factor (vascular endothelial growth factor) and its corres¬ ponding highly affine KDR-receptor on endothelial cells. KDR has been character¬ ised as a transmembranal tyrosine kinase receptor of sub-type 5 which serves as a key regulator of vascular endothelial cell development during embryogenesis and cell regeneration (Cancer and Metastasis Reviews 15: 159-163, 1996).
Furthermore, the dysfunction of that normally meticulously regulated ligand-receptor interaction results in impairment of the angiogenesis process, which is a feature of many illnesses. Particularly important is that the growth of tumours and their metastases has proved to be angiogenesis-dependent to a high degree.
A monoclonal antibody having a limited reaction pattern is already known. That antibody is directed against the extracellular domain of mouse KDR-homologous flk-1 and is capable of neutralising the VEGF stimulation of a chimeric flk-1/fms- receptor that is expressed in transfected 3T3 cells (Rockwell et al., Molecular and Cellular Differentiation, 3 (1): 91-109 (1995)).
Monoclonal antibodies that can be used for a broad spectrum of analytical assays and methods of determining human VEGF-receptor KDR in cell lysates and tissue analysates using such antibodies have not been known hitherto.
The aim of the present invention is to provide monoclonal antibodies secreted by hybridoma cell lines. The monoclonal antibodies according to the invention can be used for a broad spectrum of analytical procedures and assays, in diagnostics and as carrier molecules for therapeutic substances.
There have now been prepared monoclonal antibodies that are directed against an epitope located within extracellular domains 1 to 7 of human VEGF-receptor KDR.
The present invention relates especially to monoclonal antibodies that are directed against an epitope located within extracellular domains 6 and 7 of human VEGF- receptor KDR.
The monoclonal antibodies AM 2-7-9, AM 2-10-1 , AM 5-1-2, AM 5-10-13 and AM 2-4-1 are preferred.
The monoclonal antibodies AM 2-7-9 and AM 2-10-1 are especially preferred.
Those antibodies are directed specifically against an epitope that is located within extracellular domains 6 and 7 of human VEGF-receptor KDR.
The antibodies according to the invention exhibit a high degree of selectivity in a broad spectrum of analytical procedures and assays.
The monoclonal antibodies according to the invention have the advantage that they neither interfere with the ligand-binding domain nor activate KDR after binding.
The antibodies according to the invention are also valuable for diagnostic purposes.
For example, the monoclonal antibodies according to the invention can be used in Western blots, immunoprecipitation, ELISA, FACS analysis and in indirect immuno- fluoresence microscopy.
A further use of the monoclonal antibodies is in immunohistochemistry.
The monoclonal antibodies can also be used in screening for small agonistic and antagonistic molecules and in the detection of mutant receptor subtypes.
The monoclonal antibodies according to the invention can also be used in diagnostics, it being possible to couple them in combination with a suitable contrast- enhancing substance. The antibodies react with an epitope that is not located within the ligand-biπding side of the KDR and is therefore capable of reacting with all KDR- receptor populations.
A preferred contrast-enhancing substance that can be coupled to the antibodies is 99m technetium.
The monoclonal antibodies according to the invention can also be used in angio- genesis-dependent phenotypes, such as tumours and metastases thereof, rheuma¬ toid arthritis or psoriasis, or pathological symptoms resulting therefrom, when coupled with suitable toxic substances
Suitable toxic substances are adequately known to the person skilled in the art and are described, for example, in Byers & Baldwin, Immunology (1988), 65, 329-335 and in Blakey et al., Waldmann H. (ed) Monoclonal Antibody Therapy, Prog. Allergy Basle, Karger, (1988), vol. 45, 50-90
The invention relates also to the use of mRNA that codes for the heavy and the light chain of the antibodies according to the invention in the preparation of recombiπant "single-chain antibodies"
The invention relates also to the use of the recombmant antibodies encoded by mRNA for Western blots, immunoprecipitation, ELlSA.and FACS analysis, in indirect immunofluoresence microscopy and immuπohistochemistry and in screening for small agonistic and antagonistic molecules and in the detection of mutant receptor subtypes.
The invention relates also to a method of determining human VEGF-receptor KDR in cell lysates or tissue analysates, characterised in that
1 the monoclonal antibodies AM 2-7-9, 2-10-1 , AM 5-1 -2, AM 5-10-13 or
AM 2-4-1 , as captor antibodies, are coupled in purified form at a concen- tration of 1 - 10 μg/ml in coupling buffer on ELISA plates and then excess binding sites are blocked with blocking buffer,
2. cell or tissue analysates are prepared in a suitable lysis buffer,
3. the plates are washed in washing buffer before application of the lysates,
4. the KDR-protein to be determined in the sample is quantified using a recombiπaπt KDR-protein as standard curve,
5. the tissue and cell lysates to be analysed are then introduced into the test system,
6. incubation is carried out at room temperature for 2 hours,
7. the ELISA plates are then washed.
8. determination of the "captured" KDR is carried out by means of a polycioπa! aπti-KDR antiserum
and finally
9. detection by means of a chromσgeπic, chemiluminescent or radioactive substance is carried out.
The method can also take place in the form of a kiπase test procedure that deter¬ mines tyrosine-phosphorylated KDR.
In order to allow quantification of the test method, recombinaπt phosphorylated KDR can be used as the standard protein.
The determination of human VEGF-recsptor KDR in cell lysates or tissue analysates can be carried out both qualitatively and quantitatively using the method.
The poiyclonal anti-KDR antiserum used in step 8 of the method can be, for example, polycloπal anti-phosphotyrosine antiserum or a monoclonal anti-phospho¬ tyrosine antibody.
The detection carried out in step 9 of the method can preferably be carried out with peroxidase-labelled secondary antibodies and suitable chromogenic or chemi- lu inescent substrates.
The antiserum used can be, for example, from goats or rabbits or from sheep, rats or donkeys, either directly in the form of enzyme-labelled immunoglobulin or indirectly by means of enzyme-labelled anti-goat, anti-rabbit, anti-sheep, anti-rat or anti- donkey antibodies. Either alkaline phosphatase or peroxidase is used for that purpose.
The ELISA plates used in step 1 of the method can be antibody-coated and blocked.
The lysis buffer used in step 2 of the method contains 1-5 mM divalent ions and 1-15 % glycerol.
The lysis buffer preferably contains 1-2 mM divalent ions and 5-12 % glycerol.
A lysis buffer that contains 1.5 mM divalent ions and 10 % glycerol is especially preferred.
A preferred divalent ion is, for example, magnesium.
The washing and dilution buffers used in the method can be any buffers known to the person skilled in the art for that purpose.
Preferred washing buffers are those containing PBS with 0.05 % detergent and 0.1 % bovine serum albumin.
Preferred dilution buffers are those containing 1 % bovine serum albumin.
Description of the Figures
Fig. 1 shows a gel with the specific reaction pattern using the example of the monoclonal antibody AM 2-10-1 :
track 1 : pig aorta endothelial cells, 25 μg total cell lysate track 2: pig aorta endothelial cells, transfected with human KDR, 25 μg total cell lysate track 3 CHO cells, transfected with human KDR, 25 μg total cell lysate track 4 CHO cells, 25 μg total cell lysate track 5 human umbilical cord endothelial cells, passage 6, 25 μg total cell lysate track 6. mouse endothelial cells, 25 μg total cell lysate
Fig. 2 shows the gel for immunoprecipitation with the monoclonal antibodies AM 2-10-1 and AM 2-7-9:
track 1 5 μg AM 2-7-9 track 2 1 μg AM 2-7-9 track 3 5 μg AM 2-10-1 track 4 1 μg AM 2-10-1 track 5 isotype control, 10 μg non-specific antibody of subclass lgG1
Fig. 3 shows epitope mapping:
track 1 : soluble KDR, domains 1 and 2 track 2: soluble KDR, domains 1 to 3 track 3: soluble KDR, domains 1 to 5 track 4: soluble KDR, domains 1 to 7
The following Examples describe the preparation of monoclonal antibodies according to the invention and use thereof, but the invention is not limited to these Examples.
1. Preparation of monoclonal antibodies
Female Balb/c mice (six weeks old) are hyper-immunised either with soluble receptor (20 μg/injection) in Freund's complete adjuvant or with bacculovirus-infected SF-9 cells (10 7 cells/injection in PBS) that express the full-length human KDR receptor protein. Specific antibody titres are determined using the extracellular KDR domain by ELISA. Three days after the last immunisation the spleen is removed. The splenocytes are fused with SP2O.Ag.14 murine myeloma cells in accordance with known methods (Kδhler and Millstein, Nature 256, 495, 1975). The colonies formed are screened by means of ELISA and Western blot using the soluble extracellular KDR domain. Positive colonies are cloned three times by limited dilution, purified with protein A/Sepharose chromatography and further characterised.
2. Use in Western blots
Complete cell lysates are prepared by lysis in a suitable lysis buffer that contains the above-described concentration of divalent ions and glycerol. Equal amounts of protein are separated by means of polyacrylamide gel electrophoresis under non- reducing conditions, transferred to nitrocellulose and probed with the monoclonal antibodies AM 2-7-9 and AM 2-10-1 in accordance with procedures known perse. The visualisation of the immunoreactive bands is carried out either by means of chemiluminescence or enzymatically, using alkaline-phosphatase-labelled secondary antibodies and corresponding chromogenic substrates. Fig. 1 shows the gel of a Western blot.
Use in immunoprecipitation
Immunoprecipitations are carried out using a suitable lysis buffer (50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1% IGEPAL® CA-630, 10% (v/v) glycerol, 1.5 mM MgCI 2 , 1 mM EGTA, 5 mM EDTA, 50 mM NaF, 2 mM sodium ortho-vanadate) and complete protease inhibitors in accordance with known methods. Fig. 2 shows the gel of the immunoprecipitation.
4. Use in immunohistochemistry
Cells are cultured on sterile cover glasses until shortly before confluence and are fixed with formaldehyde (4% v/v PBS per 4 g per litre glucose) before or after incubation with 20 μg/ml of the corresponding antibody in PBS. The immunoreaction is visualised with alkaline-phosphatase-labelled secondary antibodies and fast naphthol red as chromogenic substrate.
Epitope mapping
2 μg portions of recombinant soluble KDR-receptor domains that are provided with a "myc-tag" are separated by SDS-PAGE (10%) and transferred to nitrocellulose. Then two identical films are incubated with the monoclonal antibody AM 2-10-1 or with a monoclonal anti-myc antibody. Immunocomplexes are visualised by alkaline- phosphatase-labelled secondary antibodies (see Fig. 3).