WO2002066492A2 - Human adrenomedullin-specific antibody, pharmaceutical composition containing same, therapeutic uses thereof - Google Patents
Human adrenomedullin-specific antibody, pharmaceutical composition containing same, therapeutic uses thereof Download PDFInfo
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- WO2002066492A2 WO2002066492A2 PCT/FR2002/000638 FR0200638W WO02066492A2 WO 2002066492 A2 WO2002066492 A2 WO 2002066492A2 FR 0200638 W FR0200638 W FR 0200638W WO 02066492 A2 WO02066492 A2 WO 02066492A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
Definitions
- Antibody specific for human adrenomedullin pharmaceutical composition containing it, its therapeutic applications.
- the invention relates to the treatment of cancer.
- Glioblastoma is one of the most common brain tumors in humans, and is distinguished from astrocytomas by the presence of necrosis and vascular proliferation (Brain tumors, pp 433-478, Edinburgh, Churchill Livingstone, 1995).
- the treatments currently available are unfortunately only of limited effectiveness for these tumors and the prognosis is therefore unfavorable.
- PAM peptidylglycine ⁇ -amidante monooxygenase
- Adrenomedullin is a peptide exhibiting homologies with the peptide coupled to the calcitonin gene (CGRP) (from the English "calcitonin gene-related") and has therefore been classified in the family calcitonin / CGRP / amyline (Crit. Rev. Neurobiol., 11: 167-239, 1997). AM is expressed in many tissues, including the medulla of the adrenal gland, the lungs, the kidneys, and the cardiac atrium (FEBS Lett, 352: 105-108, 1994). AM has been shown to induce a multiple response in cell cultures and animal models with modulation of cell proliferation, apoptosis and induction of angiogenesis.
- CGRP calcitonin gene
- AM has also been demonstrated in various human tumors of neural or pulmonary origin, in particular cancer of the small cell lung, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma and neuroblastoma (Endocrinology, 136: 4099-4105, 1995).
- AM could be involved in the genesis of tumors.
- Two transformed glioblastoma cell lines (T98G and A172) have been shown capable of expressing and secreting AM (Peptides, 18: 1117-1124, 1997; J. Biol. Chem., 271: 23345-23351, 1996).
- AM and its receptor have also been shown to be ubiquitously expressed during embryogenesis and carcinogenesis.
- Three receptors L1, RDC1 and a receptor related to the calcitonin CRLR receptor, having different affinities for AM, have been cloned and sequenced (J. Biol. Chem., 270:
- CRLR requires the presence of partner proteins having a single transmembrane domain and called proteins modifying receptor activity (RAMPs) (Nature (Lond.), 393: 333-339,
- CRLR CRLR-associated with RAMP1
- CRLR is in mature form, fully glycosylated and binds CGRP.
- RAMP2 or RAMP3 CRLR is in partially glycosilated immature form and can bind AM.
- CRLR also has an affinity for Pamyline.
- the invention aims to meet this need.
- a first object of the invention is to provide antibodies specific for human AM or anti-AM antibodies.
- Another subject of the invention relates to the use of antibodies specific for human AM in the treatment of cancers.
- Another subject of the invention relates to a pharmaceutical composition comprising antibodies specific for human AM intended for the treatment of cancers.
- Another subject of the invention relates to the use of specific antibodies for the preparation of a medicament intended for the treatment of cancers.
- Another object of the invention relates to the use of AM in a biological test to demonstrate compounds capable of modulating the activity of AM.
- anti-AM antibodies have an inhibitory effect on the growth of tumor cells in vitro and in vivo.
- AM has a role in the angiogenesis process and that anti-AM antibodies can be used as an anti-angiogenesis agent.
- the invention relates to an antibody, or a fragment thereof, capable of recognizing the human AM protein.
- the anti-AM antibody according to the invention can be monoclonal or polyclonal. Conventional techniques for producing an antibody can be used in the context of the invention.
- antibodies to AM can be obtained by administering the AM peptide or one or more fragments of AM carrying epitopes to cells or animals, using standard techniques.
- mice can for example be mice, goats, rabbits.
- the monoclonal antibodies can also be prepared by lymphocyte hybridization, by genetic recombination, from human stem cell banks or by the "age display” technique.
- Chimeric antibodies can also be prepared, using human monoclonal antibodies comprising variable parts of mice or other species, specific for human AM.
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising as active ingredient an antibody, or a fragment thereof, specific for the AM protein.
- composition according to the invention is in particular intended for the treatment of cancers and / or tumor cells.
- prostate cancer ovarian cancer
- the treatment of neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma will be made, by way of illustration and without limitation, of prostate cancer, ovarian cancer, the treatment of neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma.
- Antibodies or fragments thereof specific for AM, contained in the compositions according to the invention, can also be used as anti-angiogenesis agents.
- the applicants have in fact demonstrated in immunohistochemical studies carried out with an anti-Factor VIII antibody, which is a marker for endothelial cells, that tumors treated with the anti-AM antibody of the invention are generally less vascularized than the control tumors, and the vascular network shows some disorganization. The vascular surface of the control tumors is thus much greater than that of the tumors treated with the anti-AM antibody.
- composition according to the invention is preferably administered by injectable route, in particular by intravenous, subcutaneous route, or by administration in the lymphatic network.
- the amount of active ingredient to be administered depends on the degree of progress of the pathology to be treated, as well as on the age and weight of the patient. Nevertheless, the unit doses generally comprise from 1 ⁇ g to 1 g, advantageously from 10 mg to 100 mg of active principle. These unit doses are normally administered one or more times a day, preferably one to three times a day.
- the invention relates to an antibody, or a fragment thereof, capable of recognizing the human AM protein, for use as an active principle of a pharmaceutical composition intended for the treatment of cancers and / or cells tumor. More particularly, the antibody according to the invention is intended for the treatment of prostate cancer, ovarian cancer, neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma.
- the invention relates to the use of an antibody, or a fragment thereof, specific for the AM protein for the preparation of a pharmaceutical composition intended for the treatment of cancers and / or tumor cells.
- this use is intended for the treatment of prostate cancer, ovarian cancer, neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids , the ganglioneuroblastoma.
- AM thus stimulates the proliferation of HUVEC cells in a very significant way after six days (p ⁇ 0.0001) of treatment of these cells with AM.
- AM also induces the migration of HUVEC cells in a very significant way (p ⁇ 0.0001) compared to control cells not treated with AM (migration test carried out on a membrane in Boyden chambers).
- AM also induces the organization of HUVEC cells into microtubules, as in the case of treatment with VEGF (Vascular Endothelial Growth Factor), after culture of HUVEC cells on an extracellular Matrigel matrix followed by treatment with AM and / or VEGF.
- VEGF Vascular Endothelial Growth Factor
- AM finally induces in vivo, in an animal model after subcutaneous implantation of a Matrigel solution, massive infiltration of cells in Matrigel and in certain regions the formation of microvessels is observed.
- AM therefore has angiogenic properties.
- Another aspect of the invention therefore relates to the use of AM in a biological test, for the detection of compounds capable of modulating the action of AM on angiogenesis or on tumor growth.
- a test according to the invention comprises the steps consisting in a) contacting a candidate compound with cells which express the AM or respond to the AM, and b) observe the fixation, or the stimulation or the inhibition of a functional response, or compare the response of cells contacted with the candidate compound with the same cells that are not contacted with the candidate compound.
- the test of the invention using conventional techniques in themselves of expression of peptides in cell cultures and known to those skilled in the art, therefore makes it possible to search for compounds, in particular small molecules capable of having in particular an inhibitory action (antagonist) on angiogenesis induced by AM and therefore to block the development of the vascular network necessary for tumor growth, or on tumor growth itself.
- FIG. 1 represents the densitometric analysis of the expression of PAM mRNA in human glioma, glioblastomal cell lines and non-tumor brain tissue.
- the total RNA (20 ⁇ g) is separated by electrophoresis on an agarose denaturing gel (1%) - formaldehyde and transferred to a Hybond-N membrane and hybridized with a 2.2 kb PAM human cDNA probe.
- the membranes are then exposed by autoradiography for 24 h. In control, after stalling, the membranes are rehybridized with a cDNA probe produced from 18S ribosomal frog RNA.
- FIG. 1 A and B the amount of PAM mRNA is normalized to the amount of 18S ribosomal RNA.
- the results correspond to the average of four independent experiments.
- NB normal brain
- GGIV grade IV glioma
- GGII grade II glioma.
- Figure 2 shows a measure of AM mRNA expression in glioblastomal lines and in the normal brain.
- Total RNA (15 ⁇ g) prepared from glioblastoma and brain cell lines is analyzed by Northern Blot. The membranes are hybridized with a human AM cDNA probe, then after detachment, rehybridized with an 18S ribosomal cDNA control for normalization.
- the quantity of AM mRNA indicated is normalized to the quantity of 18S ribosomal RNA.
- the results correspond to the average of four independent experiments.
- Figure 3 shows the RT-PCR analysis of the expression of AM mRNA in gliomas.
- the total RNA extracted from oligodendroglioma and glioblastoma are retrotranscribed into cDNA and then analyzed by quantitative RT-PCR. The results are expressed relative to the expression of an internal control, GAPDH (glyceraldehyde-3-phosphate-dehydrogenase).
- FIG. 4 represents the inhibition of the recognition of AM radiolabelled with I 125 by the anti-AM antibody.
- Glioblastomal cells U87 are incubated for 120 min at 25 ° C in the presence of 125 -AM (60 ⁇ ci ⁇ g "1 ; 7 x 10 4 cpm per test) and increasing concentrations of the antibody. Each point is the result of three experiments.
- FIG. 5 represents the effects of AM and of the anti-AM antibody on the proliferation of cells U87 (Fig. 5A) and U373 (Fig. 5B) in vitro.
- the tumor cells are seeded at a density of 6 ⁇ 10 3 cells per well in 12-well plates in the presence of MEM medium supplemented with 2% FBS, 2 mM glutamine and antibiotics.
- AM is added at a concentration of 2 x 10 "7 M and the anti-AM antibody" AMAb2 "(12 ⁇ g / ml) in the presence or not of AM (10 ⁇ M).
- C represents the non
- the controls consist of heat denatured "AMAb2" and non-relevant IgG (Ed.1).
- six points are used for MTT measurement and six points are used for counting cells in a counter. Coulter.
- Vertical bars represent SEM. **: p ⁇ 0.007; ***: p ⁇ 0.0001.
- Figure 6 shows treatment with xenografts anti-adrenomedullin antibody to established human tumors.
- Mice not treated with AMAb2 are sacrificed between 21 and 24 days, due to the size of the tumors.
- the average tumor volume over a period of more than 70 days is shown in Figure 6.
- the sizes of the tumors in the PBS group were not different from those obtained in the non-control IgG group. Cell culture and cell proliferation tests.
- Human glioblastoma cell lines are obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and are cultured in minimal essential medium (MEM) (U373, U138, U87) or in L15 medium (SW1783 and SW1088 ), available from Life Technologies, Paris, France, in the presence of penicillin (50 U / ml), streptomycin (50 ⁇ g / ml), glutamine (1 mg / ml) and fetal calf serum (10 %). The cells are cultured in a humid atmosphere 95% air / 5% C0 2 . The culture medium is changed every two days. The amidation activity and the RNAs are prepared when the cells are in the exponential growth phase.
- MEM minimal essential medium
- SW1783 and SW1088 L15 medium
- fetal calf serum 10 fetal calf serum
- Tumor tissues from patients operated on for gliomas are studied. 5 grade II gliomas (low grade), 7 anaplastic oligodendrogliomas and 13 glioblastomas (grade IV) (according to the WHO histopathological classification) are used. Tumor samples are collected at the time of surgery and immediately stored in liquid nitrogen until the RNAs are extracted. Normal telencephalic tissue is removed from a patient operated on for epilepsy treatment. All tissue sampling protocols were performed according to the requirements of the institutional committees and with the agreement of the patients.
- RNAs The extraction of total RNAs is carried out from human gliomas, normal telencephalic tissue and cell lines using the method guanidinium isothiocyanate / phenol / chloroform (Anal. Biochem., 162: 156-159, 1987) before analysis by Northern Blot.
- the cells are detached and collected in cold PBS buffer, and prepared for an amidation test.
- IR-AM anti-AM immunostaining
- the cells are prepared according to the procedure described in FEBS Lett., 352: 105-108, 1994.
- the chromatographic characterization of IR-AM in the culture medium is carried out by High performance liquid chromatography in reverse phase, using a ⁇ Bondapak C18 column (3.9 x 300 mm; Waters).
- the conditioned medium (15 ml) is extracted using Sep-Pak C18 cartridges.
- the extract is reconstituted with a solution containing 0.1% (vol / vol) of trifluoroacetic acid and loaded on the column.
- the analysis is carried out with a linear gradient of acetonitrile containing 0.1% (vol / vol) of trifluoroacetic acid, from 10 to 60% at a speed of 1 ml / min / fraction for 50 min. Each fraction (1 ml) is collected, dried and analyzed. Quantitative RT-PCR.
- a real-time quantitative RT-PCR (polymerase chain reaction) method is used to measure variations in the copy number of AM or GAPDH.
- Total DNA free RNA (2 ⁇ g) is transcribed into complementary DNA (cDNA) in the presence of a ⁇ g of hexamers (Pharmacia Biotech, Orsay, France) and of reverse transcriptase MMLV (from the English "Moloney leukemia virus "), as described by Life Technologies Inc., Paris, France.
- AM and human GAPDH mRNA is amplified (AM: primer sense 5'-TGCCCAGACCCTTATTCGG-3 'and antisense primer 5'-AGTTGTTCATGCTCTGGCGG-3'; GAPDH: primer sense 5'- CAAATTCCATGGCACCGTC-3 'and antisense primer 5'-
- the Taq Man probes for AM and GAPDH are 5'- ACATGAAGGGTGCCTCTCGAAGCCC-3 'and 5'-
- the amplification mixture contains the cDNA produced from 50-150 ng of total RNA, 0.2 ⁇ M of primer, and 0.1 ⁇ M of Taq Man probe in 50 m of NaCl and 5 mM of Mg 2+ .
- a two-step PCR is performed at 35 cycles. The denaturation is done at 94 ° C for 20 sec, and the fixing of the primers on the matrix / extension at 60 ° C for 30 sec.
- the reaction produces a PCR product of 115 base pairs (bp) for AM and 101 bp for GAPDH.
- AM mRNA levels are normalized to GAPDH mRNA levels in the same reaction.
- RNAs are produced by in vitro transcription from the linearized cDNA matrices of AM and GAPDH by the T7 or T3 polymerases then retrotranscribed into cDNA.
- AM and GAPDH probes labeled with fluorochromes under the experimental conditions defined above, a linear relationship between the concentration of RNA retrotranscribed into cDNA and the fluorescence signal ( ⁇ RQ) of the RNAs of AM and GAPDH in 1- 250 pg target DNA is obtained.
- ⁇ RQ values are determined for the two genes, and the results are expressed in fg of AM per pg of GAPDH.
- Primers as defined in Table 1 appended to this patent application are selected to be specific for CRLR, RAMP2 and RAMP3.
- the PCR reactions initiated by the specific primer sets for CRLR, RAMP2 and RAMP3 are carried out with cDNA produced from glioma cell lines and from a sample taken from a human glioma.
- the cycle parameters are as follows: Initial denaturation stage: 94 ° C., 5 min,
- New Zealand female rabbits are immunized three times subcutaneously with human AM (Bachem) (120 ⁇ g AM equivalent per injection: the first dose in a complete Freund's adjuvant, the last two in an incomplete Freund's adjuvant ).
- the sera are selected for their anti-AM activity, then affinity purified on a protein A Sepharose column (Amersham Pharmacia Biotech).
- Glioblastoma cells are cultured in 24-well plates until confluence (1 ⁇ 10 5 cells / well) and deprived in serum for 24 h after washing with PBS buffer without Ca + added with 0.2% bovine serum albumin (BSA) and Bacitracin (100 mg / ml, Sigma), the cells are incubated at 25 ° C for 120 min with the radioactive marker (I 125 ) in the presence or absence of an excess (10 "6 M) of Non-radiolabelled AM
- the synthetic iodine labeling of human AM (Amh) is carried out by the chloranine T method and the product is purified by reverse phase high performance liquid chromatography.
- Monoiodized Amh (I 125 ) (SA, 350 Ci / mmol) is used in the experiments.
- the cells are incubated with the radiolabeled marker and increasing concentrations of anti-AM antibodies.
- the cells are washed with Cold PBS containing 0.2% BSA, solubilized with 0.2 M sodium hydroxide, and the radioactivity retained is measured by a ⁇ spectrometer.
- the specific binding is obtained by subtracting the measurement of non-specific recognition obtained in the presence of unmarked AMh in excess of the measurement of total recognition.
- the data represents the average of three experiments, each performed three times. Animals.
- mice (Nu / Nu) from 4 to 5 weeks (Janvier, Laval Le Genest, France), are placed in sterile cages under closed laminar flow, at controlled temperature and with periods of 12 h of light and 12 h of darkness, and fed and quenched ad libitum. In vivo anti-tumor therapy.
- U87 cells (3 x 10 6 cells / mouse) in exponential growth phase are injected subcutaneously into the flanks of athymic mice. After two weeks, the tumors have grown to a volume of approximately 1350 +/- 170 mm 3 . The treatment is started after 14 days.
- the anti-AM AMAb2 antibody (200 ⁇ g of purified IgG) is injected into the tumor (20 mice) in a volume of 0.2 ml, twice a week.
- a negative control a rabbit anti-IgG directed against the peptide derived from the rat endothelin precursor or the phosphate buffer (PBS) alone are used. Each control group has seven mice.
- Each mouse within groups treated with immunoglobulin receives a total of 400 ⁇ g of immunoglobulin per week for the duration of treatment.
- Tumors are measured twice a week during treatment.
- Statistical analyzes are performed using the ANOVA method followed by Fisher's significant difference test (Statview 512, Brain Power Inc., Calabasas, CA, USA). A value of p ⁇ 0.05 is considered significant.
- RNA from human glioma, human glioma cell lines and normal brain tissue is prepared to analyze the level of PAM expression.
- a 2.2 kilobase (kb) probe the transcript corresponding to PAM is highlighted by Northern Blot at 4 kb.
- a high level of expression is demonstrated in the human glioma (Fig. 1A) and the cell lines (Fig. 1B) in comparison with normal tissues.
- a second labeling is carried out with the cDNA probe prepared from 18S rRNA.
- PAM mRNA levels are 9 to 15 times higher in malignant glioblastomas and 4 to 6 times higher in low-grade gliomas than in normal brain tissue.
- the majority of cell lines derived from human glioblastomas consistently express high levels of PAM mRNA (Fig. 1 B).
- PAM activity in cell lines The amidation activity of the extracts from each cell line is tested using an ⁇ -N-acetyl-Tyr-Val-Gly substrate at pH 5.5.
- U373 and SW1783 high-grade glioma cell lines have higher PAM activity levels (77 +/- 4 pmol / mg protein / h) than U87, U 138 and SW1088 lines (55 +/- 4.3 pmol / mg protein / h).
- the expression of PAM in both tumors and gliomal lines demonstrates their ability to produce ⁇ -amide peptides. Expression of AM mRNA in cell lines.
- AM has a very high level of expression.
- the level of expression of the AM mRNA is analyzed by Northern Blot on gliomal lines and normal human tissues (Biochem. Biophys. Res. Commun., 194: 720-725, 1993).
- the size of the transcript is approximately 1.6 kb.
- the amount of AM mRNA is then normalized with respect to the amount of 18S ribosomal RNA (Fig. 2). All the gliomal cell lines tested express the AM mRNA, while no expression is detected in non-tumor tissues.
- Anti-AM immunostaining is positive in cell extracts and the culture medium. All gliomal lines therefore produce and secrete AM.
- the quantity of AM obtained is indicated in Table 2 in the appendix to this patent application.
- Total RNA is prepared from samples of 25 glioma tumors comprising 13 high degree (IV) gliomas, 5 low degree (II) gliomas and 7 anaplastic oligodendrogliomas.
- the levels of expression of AM mRNA are indicated in FIG. 3.
- the quantification of AM mRNA transcripts reveals a higher level of expression of AM mRNA in glioblastomas, compared to the oligodendrogliomas of low degree or anaplastic.
- the polyclonal anti-AM antibody (serum or purified IgG) clearly recognizes full-size AM but does not show any cross-reactivity with peptides related to AM, as it appears in Table 3 appearing in the appendix to the present patent application. Calcitonin, CGRP ⁇ . 3) CGRP 8 ⁇ 7 and the amylin only show a non-significant fixation with the antibody. In addition, no cross-reaction with the peptides ACTH (human 1-24), endotheline-1 (human), AVP (human), CRF (human), TRH, substance P, ANF (human) is observed.
- the ability of the antibody to block the binding of AM-I 125 to its receptor in U87 cells is tested (Fig. 4).
- the antibody blocks the receptor-AM interaction in a dose-dependent manner. Effects of AM and anti-AM antibody on the proliferation of U87 and U373 cells in vitro.
- U87 and U373 cells are cultured in the presence of AM 2 x 10 "7 M, and the effects of the peptide on cell proliferation are examined by an MTT method.
- 5A and 5B show that AM at a concentration of 2 x 10 "7 M, stimulates the proliferation of U87 cells by 15% (p ⁇ 0.02) and U373 by 20% (p
- a polyclonal anti-AM antibody (purified IgG) is tested for its inhibitory effect on the proliferation of U87 and U373 cells cultured in the presence of antibodies. After 7 days of treatment, the antibody at a concentration of 12 ⁇ g / ml inhibits the proliferation of U87 and U373 by 33% (p ⁇ 0.002) and 50% (p ⁇ 0.001) respectively (Fig. 5A and 5B).
- mice with tumors Effects of the anti-AM antibody in vivo on mice with tumors.
- the ability of the anti-AM antibody to inhibit growth in vivo was tested on U87 cells xenografted in mice.
- the cell line produces a palpable tumor mass (506 +/- 70 mm 3 ).
- 14th th day (1350 +/- 170 mm 3) tumors are subjected to three treatments: PBS buffer, non relevante specificity of IgG anti-AM antibody (purified IgG).
- FIG. 6 shows the growth curves of tumors of mice carrying a human glioblastoma U87, which are treated every three days with the anti-AM antibody, or the non-relevant IgG control.
- the treatment is administered by intra-tumor injection and the growth is determined as a function of the tumor volume over time. After 21 days of treatment with the antibody at a dose of 200 ⁇ g twice a week, the average volume of the tumor is reduced to 646 +/-
- the reference corresponds to the Genbank number.
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Abstract
Description
Anticorps spécifique de l'adrénomedulline humaine, composition pharmaceutique le contenant, ses applications thérapeutiques. Antibody specific for human adrenomedullin, pharmaceutical composition containing it, its therapeutic applications.
L'invention se rapporte au traitement du cancer. Le glioblastome est l'une des tumeurs du cerveau la plus courante chez l'homme, et se distingue des astrocytomes par la présence de nécroses et de proliférations vasculaires (Brain tumors, pp 433-478, Edinburgh, Churchill Livingstone, 1995). Les traitements actuellement disponibles ne sont malheureusement que d'une efficacité limitée pour ces tumeurs et le pronostic est par conséquent défavorable.The invention relates to the treatment of cancer. Glioblastoma is one of the most common brain tumors in humans, and is distinguished from astrocytomas by the presence of necrosis and vascular proliferation (Brain tumors, pp 433-478, Edinburgh, Churchill Livingstone, 1995). The treatments currently available are unfortunately only of limited effectiveness for these tumors and the prognosis is therefore unfavorable.
On sait que les facteurs de croissance régulent la prolifération et la différentiation cellulaires et sont directement impliqués dans la transformation néoplasique (Ann. Intern. Med. 117 : 408-414, 1992). L'une des caractéristiques communes de beaucoup de ces peptides (hormones, facteurs de croissance, enzymes), essentielle à leur fonction dans la communication intracellulaire, est la présence d'un groupe α-amide à l'extrémité carboxyle (Trends Biochem. Sci., 16 : 112-115, 1991).It is known that growth factors regulate cell proliferation and differentiation and are directly involved in neoplastic transformation (Ann. Intern. Med. 117: 408-414, 1992). One of the common characteristics of many of these peptides (hormones, growth factors, enzymes), essential to their function in intracellular communication, is the presence of an α-amide group at the carboxyl end (Trends Biochem. Sci ., 16: 112-115, 1991).
Un seul complexe enzymatique, la peptidylglycine α-amidante monooxygénase (PAM) est responsable de l'α-amidation de ces peptides et hormones (Annu. Rev. Neurosci. 15 : 57-85, 1992). Une activité PAM a ainsi été trouvée dans des tumeurs endocrines sécrétant des peptides α-amidés, notamment dans le carcinome thyroïdien médullaire, le phéochromocytome (Mol. Cell. Endochnology, 79 : 53-63, 1991 ), les tumeurs pancréatiques sécrétant le peptide intestinal vasoactif (VIP) (Clinical Endocrinology, 33 : 467-480, 1990), et chez l'homme dans des tumeurs hypophysaires (Metabolism, 34 : 1044-1052, 1985).A single enzyme complex, peptidylglycine α-amidante monooxygenase (PAM) is responsible for the α-amidation of these peptides and hormones (Annu. Rev. Neurosci. 15: 57-85, 1992). PAM activity has thus been found in endocrine tumors secreting α-amide peptides, in particular in medullary thyroid carcinoma, pheochromocytoma (Mol. Cell. Endochnology, 79: 53-63, 1991), pancreatic tumors secreting intestinal peptide vasoactive (VIP) (Clinical Endocrinology, 33: 467-480, 1990), and in humans in pituitary tumors (Metabolism, 34: 1044-1052, 1985).
L'adrénomedulline (AM) est un peptide présentant des homologies avec le peptide couplé au gène de la calcitonine (CGRP) (de l'anglais "calcitonin gene- related") et a été classée de ce fait dans la famille calcitonine / CGRP / amyline (Crit. Rev . Neurobiol., 11 : 167-239, 1997). AM est exprimée dans de nombreux tissus, notamment la medulla de la glande surrénale, les poumons, les reins, et l'atrium cardiaque (FEBS Lett, 352 : 105-108, 1994). AM a été montré induisant une réponse multiple sur cultures cellulaires et modèles animaux avec modulation de la prolifération cellulaire, de l'apoptose et induction de l'angiogenèse.Adrenomedullin (AM) is a peptide exhibiting homologies with the peptide coupled to the calcitonin gene (CGRP) (from the English "calcitonin gene-related") and has therefore been classified in the family calcitonin / CGRP / amyline (Crit. Rev. Neurobiol., 11: 167-239, 1997). AM is expressed in many tissues, including the medulla of the adrenal gland, the lungs, the kidneys, and the cardiac atrium (FEBS Lett, 352: 105-108, 1994). AM has been shown to induce a multiple response in cell cultures and animal models with modulation of cell proliferation, apoptosis and induction of angiogenesis.
L'expression de AM a également été mise en évidence dans différentes tumeurs humaines d'origine neurale ou pulmonaire, notamment le cancer du poumon à petites cellules, l'adénocarcinome, le carcinome bronchoalvéolaire, le carcinome de cellules squameuses, les carcinoïdes pulmonaires, le ganglioneuroblastome et le neuroblastome (Endocrinology, 136 : 4099-4105, 1995).The expression of AM has also been demonstrated in various human tumors of neural or pulmonary origin, in particular cancer of the small cell lung, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma and neuroblastoma (Endocrinology, 136: 4099-4105, 1995).
AM pourrait être impliquée dans la genèse des tumeurs. Deux lignées cellulaires transformées de glioblastome (T98G et A172) ont été montrées capables d'exprimer et de sécréter AM (Peptides, 18 : 1117-1124, 1997; J. Biol. Chem., 271 : 23345-23351 , 1996).AM could be involved in the genesis of tumors. Two transformed glioblastoma cell lines (T98G and A172) have been shown capable of expressing and secreting AM (Peptides, 18: 1117-1124, 1997; J. Biol. Chem., 271: 23345-23351, 1996).
Il a été également montré que AM et son récepteur (AM-R) sont exprimés de façon ubiquitaire pendant l'embryogenèse et la carcinogenèse. Trois récepteurs (L1 , RDC1 et un récepteur apparenté au récepteur de la calcitonine CRLR), présentant différentes affinités pour AM, ont été clones et séquences (J. Biol. Chem., 270 :AM and its receptor (AM-R) have also been shown to be ubiquitously expressed during embryogenesis and carcinogenesis. Three receptors (L1, RDC1 and a receptor related to the calcitonin CRLR receptor), having different affinities for AM, have been cloned and sequenced (J. Biol. Chem., 270:
24344-25347, 1995). Ils appartiennent tous à la famille des récepteurs à 7 domaines transmembranaires couplés aux protéines G. CRLR nécessite la présence de protéines partenaires possédant un seul domaine transmembranaire et appelées protéines modifiant l'activité de récepteur (RAMPs) (Nature (Lond.), 393 : 333-339,24344-25347, 1995). They all belong to the family of receptors with 7 transmembrane domains coupled to proteins G. CRLR requires the presence of partner proteins having a single transmembrane domain and called proteins modifying receptor activity (RAMPs) (Nature (Lond.), 393: 333-339,
1998). L'association du CRLR et des protéines RAMP spécifie la nature des ligands liés à ce complexe. Associé à RAMP1 , CRLR est sous forme mature, pleinement glycosylé et lie le CGRP. Associé à RAMP2 ou RAMP3, CRLR est sous forme immature partiellement glycosilé et peut lier l'AM. Dans le cas d'une association avec RAMP3, CRLR présente aussi une affinité pour Pamyline.1998). The association of CRLR and RAMP proteins specifies the nature of the ligands linked to this complex. Associated with RAMP1, CRLR is in mature form, fully glycosylated and binds CGRP. Associated with RAMP2 or RAMP3, CRLR is in partially glycosilated immature form and can bind AM. In the case of an association with RAMP3, CRLR also has an affinity for Pamyline.
Bien qu'une sur-expression de AM ait été mise en évidence dans plusieurs tumeurs (J. Clin. Endocrinol. Metabol., 80 : 1750-1752, 1995), les effets réels de AM sur la genèse des tumeurs restent inconnus.Although over-expression of AM has been demonstrated in several tumors (J. Clin. Endocrinol. Metabol., 80: 1750-1752, 1995), the actual effects of AM on the genesis of tumors remain unknown.
Il existe donc toujours aujourd'hui un besoin en des traitements capables de ralentir ou de stopper le développement de tumeurs cancéreuses.There is therefore still a need today for treatments capable of slowing down or stopping the development of cancerous tumors.
Il existe toujours également un besoin en des méthodes utiles pour mettre en évidence des composés utilisables dans le traitement de cancers.There also still exists a need for useful methods for detecting compounds which can be used in the treatment of cancers.
L'invention vise à répondre à ce besoin.The invention aims to meet this need.
Un premier objet de l'invention est de fournir des anticorps spécifiques de AM humaine ou anticorps anti-AM.A first object of the invention is to provide antibodies specific for human AM or anti-AM antibodies.
Un autre objet de l'invention concerne l'utilisation d'anticorps spécifiques de AM humaine dans le traitement de cancers.Another subject of the invention relates to the use of antibodies specific for human AM in the treatment of cancers.
Un autre objet de l'invention concerne une composition pharmaceutique comprenant des anticorps spécifiques de AM humaine destinée au traitement de cancers. Un autre objet de l'invention concerne l'utilisation d'anticorps spécifiques pour la préparation d'un médicament destiné au traitement de cancers.Another subject of the invention relates to a pharmaceutical composition comprising antibodies specific for human AM intended for the treatment of cancers. Another subject of the invention relates to the use of specific antibodies for the preparation of a medicament intended for the treatment of cancers.
Un autre objet de l'invention concerne l'utilisation de AM dans un test biologique pour mettre en évidence des composés capables de moduler l'activité de AM.Another object of the invention relates to the use of AM in a biological test to demonstrate compounds capable of modulating the activity of AM.
Selon l'invention, il a été trouvé que les anticorps anti-AM possèdent un effet inhibiteur sur la croissance de cellules tumorales in vitro et in vivo.According to the invention, it has been found that anti-AM antibodies have an inhibitory effect on the growth of tumor cells in vitro and in vivo.
Selon l'invention, il a été trouvé également que AM possède un rôle dans le processus d'angiogénèse et que les anticorps anti-AM peuvent être utilisés n tant qu'agent anti-angiogénèse.According to the invention, it has also been found that AM has a role in the angiogenesis process and that anti-AM antibodies can be used as an anti-angiogenesis agent.
Selon un premier aspect, l'invention concerne un anticorps, ou un fragment de celui-ci, capable de reconnaître la protéine AM humaine.According to a first aspect, the invention relates to an antibody, or a fragment thereof, capable of recognizing the human AM protein.
L'anticorps anti-AM selon l'invention peut être monoclonal ou polyclonal. Les techniques classiques de production d'un anticorps peuvent être utilisées dans le cadre de l'invention.The anti-AM antibody according to the invention can be monoclonal or polyclonal. Conventional techniques for producing an antibody can be used in the context of the invention.
Par exemple, des anticorps dirigés contre AM peuvent être obtenus en administrant le peptide AM ou un ou plusieurs fragments de AM portant des épitopes à des cellules ou des animaux, en utilisant les techniques classiques.For example, antibodies to AM can be obtained by administering the AM peptide or one or more fragments of AM carrying epitopes to cells or animals, using standard techniques.
Le choix des animaux utilisés pour la production d'anticorps n'est pas limitatif en soi. Il peut s'agir par exemple de souris, de chèvre, de lapin.The choice of animals used for the production of antibodies is not limiting in itself. It can for example be mice, goats, rabbits.
Pour la préparation d'anticorps monoclonaux, les techniques fournissant des anticorps produits par des cultures de lignées cellulaires en continu peuvent être utilisées. Des exemples de techniques comprennent la technique des hybridomes (Kohler, G. , Milstein, C, Nature, 256 : 495-497, 1975), la technique des trioma, la technique des hybridomes de cellules B humaines (Kozbor et. al., Immunology Today, 4 : 72, 1983) et la technique des hybridomes EBV (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).For the preparation of monoclonal antibodies, techniques providing antibodies produced by cultures of cell lines continuously can be used. Examples of techniques include the hybridoma technique (Kohler, G., Milstein, C, Nature, 256: 495-497, 1975), the trioma technique, the human B cell hybridoma technique (Kozbor et. Al., Immunology Today, 4: 72, 1983) and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).
On peut également préparer les anticorps monoclonaux par hybridation lymphocytaire, par recombinaison génétique, à partir de banques de cellules souches humaines ou par la technique de "p age display".The monoclonal antibodies can also be prepared by lymphocyte hybridization, by genetic recombination, from human stem cell banks or by the "age display" technique.
Des anticorps chimériques peuvent également être préparés, en utilisant des anticorps monoclonaux humains comportant des parties variables de souris ou d'autres espèces, spécifiques de l'AM humaine.Chimeric antibodies can also be prepared, using human monoclonal antibodies comprising variable parts of mice or other species, specific for human AM.
Les techniques pour la production d'anticorps à chaîne unique (U.S. Patent No. 4,946,778) peuvent également être adaptées pour produire les anticorps de l'invention. Egalement, les souris transgéniques ou d'autres organismes incluant des mammifères différents, peuvent être utilisées.Techniques for the production of single chain antibodies (US Patent No. 4,946,778) can also be adapted to produce the antibodies of the invention. Also, transgenic mice or other organisms including different mammals can be used.
Selon un autre aspect, l'invention concerne une composition pharmaceutique comprenant en tant que principe actif un anticorps, ou un fragment de celui-ci, spécifique de la protéine AM.According to another aspect, the invention relates to a pharmaceutical composition comprising as active ingredient an antibody, or a fragment thereof, specific for the AM protein.
La composition selon l'invention est en particulier destinée au traitement de cancers et/ou de cellules tumorales.The composition according to the invention is in particular intended for the treatment of cancers and / or tumor cells.
On citera à titre illustratif et non limitatif le cancer de la prostate, le cancer des ovaires, le traitement des neuroblastomes, des glioblastomes, le cancer du poumon, l'adenocarcinome, le carcinome bronchoalvéolaire, le carcinome de cellules squameuses, les carcinoïdes pulmonaires, le ganglioneuroblastome.Mention will be made, by way of illustration and without limitation, of prostate cancer, ovarian cancer, the treatment of neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma.
Les anticorps ou fragments de ceux-ci spécifiques de l'AM, contenus dans les compositions selon l'invention, peuvent également être utilisés en tant qu'agents anti-angiogénèse. Les demandeurs ont en effet mis en évidence dans des études immunohistochimiques réalisées avec un anticorps anti-Facteur VIII, qui est un marqueur de cellules endothéliales, que des tumeurs traitées avec l'anticorps anti- AM de l'invention sont globalement moins vascularisées que les tumeurs contrôles, et que le réseau vasculaire présente une certaine désorganisation. La surface vasculaire des tumeurs contrôles est ainsi très supérieure à celle des tumeurs traitées avec l'anticorps anti-AM. Ces résultats montrent l'implication originale de l'AM dans la mise en place et le maintien d'un réseau tumoral et peritumoral stable et fonctionnel.Antibodies or fragments thereof specific for AM, contained in the compositions according to the invention, can also be used as anti-angiogenesis agents. The applicants have in fact demonstrated in immunohistochemical studies carried out with an anti-Factor VIII antibody, which is a marker for endothelial cells, that tumors treated with the anti-AM antibody of the invention are generally less vascularized than the control tumors, and the vascular network shows some disorganization. The vascular surface of the control tumors is thus much greater than that of the tumors treated with the anti-AM antibody. These results show the original involvement of AM in the establishment and maintenance of a stable and functional tumor and peritumoral network.
La composition selon l'invention est de préférence administrée par voie injectable, notamment par voie intra-veineuse, sous-cutanée, ou par administration dans le réseau lymphatique.The composition according to the invention is preferably administered by injectable route, in particular by intravenous, subcutaneous route, or by administration in the lymphatic network.
La quantité de principe actif à administrer dépend du degré d'avancement de la pathologie à traiter, ainsi que de l'âge et du poids du patient. Néanmoins, les doses unitaires comprennent généralement de 1 μg à 1 g, avantageusement de 10 mg à 100 mg de principe actif. Ces doses unitaires sont administrées normalement en une ou plusieurs fois par jour, de préférence de une à trois fois par jour.The amount of active ingredient to be administered depends on the degree of progress of the pathology to be treated, as well as on the age and weight of the patient. Nevertheless, the unit doses generally comprise from 1 μg to 1 g, advantageously from 10 mg to 100 mg of active principle. These unit doses are normally administered one or more times a day, preferably one to three times a day.
Selon un autre aspect, l'invention concerne un anticorps, ou un fragment de celui-ci, capable de reconnaître la protéine AM humaine, pour utilisation en tant que principe actif d'une composition pharmaceutique destinée au traitement de cancers et/ou de cellules tumorales. Plus particulièrement, l'anticorps selon l'invention est destiné au traitement du cancer de la prostate, du cancer des ovaires, des neuroblastomes, des glioblastomes, le cancer du poumon, l'adenocarcinome, le carcinome bronchoalvéolaire, le carcinome de cellules squameuses, les carcinoïdes pulmonaires, le ganglioneuroblastome.According to another aspect, the invention relates to an antibody, or a fragment thereof, capable of recognizing the human AM protein, for use as an active principle of a pharmaceutical composition intended for the treatment of cancers and / or cells tumor. More particularly, the antibody according to the invention is intended for the treatment of prostate cancer, ovarian cancer, neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids, ganglioneuroblastoma.
Selon encore un autre aspect, l'invention concerne l'utilisation d'un anticorps, ou d'un fragment de celui-ci, spécifique de la protéine AM pour la préparation d'une composition pharmaceutique destinée au traitement de cancers et/ou de cellules tumorales. Selon l'invention, cette utilisation est destinée au traitement du cancer de la prostate, du cancer des ovaires, des neuroblastomes, des glioblastomes, le cancer du poumon, l'adenocarcinome, le carcinome bronchoalvéolaire, le carcinome de cellules squameuses, les carcinoïdes pulmonaires, le ganglioneuroblastome.According to yet another aspect, the invention relates to the use of an antibody, or a fragment thereof, specific for the AM protein for the preparation of a pharmaceutical composition intended for the treatment of cancers and / or tumor cells. According to the invention, this use is intended for the treatment of prostate cancer, ovarian cancer, neuroblastomas, glioblastomas, lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, pulmonary carcinoids , the ganglioneuroblastoma.
L'utilité des anticorps de l'invention en tant qu'agents anti-angiogénèse résulte de la mise en évidence par les demandeurs du rôle de l'AM dans le processus d'angiogénèse.The usefulness of the antibodies of the invention as anti-angiogenesis agents results from the demonstration by the applicants of the role of AM in the process of angiogenesis.
Cette implication de l'AM est démontrée à plusieurs niveaux, en utilisant des cellules endothéliales ombilicales humaines (HUVEC).This involvement of AM is demonstrated at several levels, using human umbilical endothelial cells (HUVEC).
L'AM stimule ainsi la prolifération des cellules HUVEC d'une façon très significative après six jours (p < 0,0001) de traitement de ces cellules par AM. L'AM induit également la migration des cellules HUVEC d'une manière très significative (p < 0,0001 ) par rapport aux cellules contrôles non traitées par AM (test de migration effectué sur membrane dans des chambres Boyden). Les effets des cellules traitées avec AM sont plus importants que ceux provoqués par le FGF (Fibroblast Growth Factor, où p = 0,0092).AM thus stimulates the proliferation of HUVEC cells in a very significant way after six days (p <0.0001) of treatment of these cells with AM. AM also induces the migration of HUVEC cells in a very significant way (p <0.0001) compared to control cells not treated with AM (migration test carried out on a membrane in Boyden chambers). The effects of cells treated with AM are greater than those caused by FGF (Fibroblast Growth Factor, where p = 0.0092).
Un traitement réalisé avec les deux facteurs montre un effet additif suggérant l'activation de deux voies indépendantes qui seraient à l'origine de la migration (p = 0,0002).A treatment carried out with the two factors shows an additive effect suggesting the activation of two independent pathways which would be at the origin of the migration (p = 0.0002).
L'AM induit également l'organisation des cellules HUVEC en microtubules, comme dans les cas de traitement avec le VEGF (Vascular Endothelial Growth Factor), après culture des cellules HUVEC sur une matrice extracellulaire Matrigel suivie d'un traitement avec l'AM et / ou le VEGF.AM also induces the organization of HUVEC cells into microtubules, as in the case of treatment with VEGF (Vascular Endothelial Growth Factor), after culture of HUVEC cells on an extracellular Matrigel matrix followed by treatment with AM and / or VEGF.
L'AM induit enfin in vivo, dans un modèle animal après implantation sous cutanée d'une solution de Matrigel, une infiltration massive des cellules dans le Matrigel et dans certaines régions la formation de microvaisseaux est observée. L'AM possède par conséquent des propriétés angiogéniques. L'utilisation d'anticorps anti-AM, qui inhibent l'interaction de l'AM avec ses récepteurs et, par conséquent, tout effet biologique de l'AM, bloque ainsi la mise en place d'un réseau vasculaire stable et fonctionnel nécessaire à la croissance tumorale. Un autre aspect de l'invention concerne donc l'utilisation de l'AM dans un test biologique, pour la mise en évidence de composés capables de moduler l'action de l'AM sur l'angiogénèse ou sur la croissance tumorale.AM finally induces in vivo, in an animal model after subcutaneous implantation of a Matrigel solution, massive infiltration of cells in Matrigel and in certain regions the formation of microvessels is observed. AM therefore has angiogenic properties. The use of anti-AM antibodies, which inhibit the interaction of AM with its receptors and, consequently, any biological effect of AM, thus blocks the establishment of a necessary stable and functional vascular network. to tumor growth. Another aspect of the invention therefore relates to the use of AM in a biological test, for the detection of compounds capable of modulating the action of AM on angiogenesis or on tumor growth.
Par exemple, un test selon l'invention comprend les étapes consistant à a) mettre en contact un composé candidat avec des cellules qui expriment l'AM ou répondent à l'AM, et b) observer la fixation, ou la stimulation ou l'inhibition d'une réponse fonctionnelle, ou comparer la réponse des cellules mises en contact avec le composé candidat avec les mêmes cellules qui ne sont pas mises en contact avec le composé candidat. Le test de l'invention, en utilisant des techniques classiques en elles-mêmes d'expression de peptides dans des cultures cellulaires et connues de l'homme du métier, permet donc de rechercher des composés, en particulier des petites molécules capables d'avoir notamment une action inhibitrice (antagoniste) sur l'angiogénèse induite par l'AM et donc de bloquer le développement du réseau vasculaire nécessaire à la croissance tumorale, ou sur la croissance tumorale elle- même.For example, a test according to the invention comprises the steps consisting in a) contacting a candidate compound with cells which express the AM or respond to the AM, and b) observe the fixation, or the stimulation or the inhibition of a functional response, or compare the response of cells contacted with the candidate compound with the same cells that are not contacted with the candidate compound. The test of the invention, using conventional techniques in themselves of expression of peptides in cell cultures and known to those skilled in the art, therefore makes it possible to search for compounds, in particular small molecules capable of having in particular an inhibitory action (antagonist) on angiogenesis induced by AM and therefore to block the development of the vascular network necessary for tumor growth, or on tumor growth itself.
Des caractéristiques et avantages supplémentaires de l'invention apparaîtront dans la description détaillée qui suit, faite en relation avec les figures dans lesquelles : La figure 1 représente l'analyse densitométrique de l'expression de l'ARNm de PAM dans le gliome humain, les lignées cellulaires glioblastomales et les tissus de cerveau non tumoral. L'ARN total (20 μg) est séparé par électrophorèse sur gel dénaturant agarose (1 %) - formaldéhyde et transféré sur une membrane Hybond-N et hybrides avec une sonde d'ADNc humain de PAM de 2,2 kb. Les membranes sont ensuite exposées en autoradiographie pendant 24 h. En contrôle, après décrochage, les membranes sont réhybridées avec une sonde d'ADNc produite à partir d'ARN ribosomal 18S de grenouille. Sur la figure 1 A et B, la quantité d'ARNm de PAM est normalisée à la quantité d'ARN ribosomal 18S. Les résultats correspondent à la moyenne de quatre expériences indépendantes. NB : cerveau normal; GGIV : gliome de degré IV; GGII : gliome de degré II. La figure 2 représente une mesure de l'expression de l'ARNm de AM dans des lignées glioblastomales et dans le cerveau normal. L'ARN total (15 μg) préparé à partir de lignées cellulaires de glioblastomes et de cerveau est analysé par Northern Blot. Les membranes sont hybridées avec une sonde d'ADNc de AM humain, puis après décrochage, réhybridées avec un contrôle ADNc 18S ribosomal pour la normalisation. Sur la figure 2, la quantité d'ARNm de AM indiquée est normalisée à la quantité d'ARN ribosomal 18S. Les résultats correspondent à la moyenne de quatre expériences indépendantes.Additional characteristics and advantages of the invention will appear in the detailed description which follows, given in relation to the figures in which: FIG. 1 represents the densitometric analysis of the expression of PAM mRNA in human glioma, glioblastomal cell lines and non-tumor brain tissue. The total RNA (20 μg) is separated by electrophoresis on an agarose denaturing gel (1%) - formaldehyde and transferred to a Hybond-N membrane and hybridized with a 2.2 kb PAM human cDNA probe. The membranes are then exposed by autoradiography for 24 h. In control, after stalling, the membranes are rehybridized with a cDNA probe produced from 18S ribosomal frog RNA. In FIG. 1 A and B, the amount of PAM mRNA is normalized to the amount of 18S ribosomal RNA. The results correspond to the average of four independent experiments. NB: normal brain; GGIV: grade IV glioma; GGII: grade II glioma. Figure 2 shows a measure of AM mRNA expression in glioblastomal lines and in the normal brain. Total RNA (15 μg) prepared from glioblastoma and brain cell lines is analyzed by Northern Blot. The membranes are hybridized with a human AM cDNA probe, then after detachment, rehybridized with an 18S ribosomal cDNA control for normalization. In FIG. 2, the quantity of AM mRNA indicated is normalized to the quantity of 18S ribosomal RNA. The results correspond to the average of four independent experiments.
La figure 3 représente l'analyse par RT-PCR de l'expression de l'ARNm de AM dans les gliomes. Les ARN totaux extraits d'oligodendrogliome et de glioblastome sont rétrotranscrits en ADNc puis analysés par RT-PCR quantitative. Les résultats sont exprimés par rapport à l'expression d'un contrôle interne, la GAPDH (glycéraldéhyde-3-phosphate-déshydrogénase).Figure 3 shows the RT-PCR analysis of the expression of AM mRNA in gliomas. The total RNA extracted from oligodendroglioma and glioblastoma are retrotranscribed into cDNA and then analyzed by quantitative RT-PCR. The results are expressed relative to the expression of an internal control, GAPDH (glyceraldehyde-3-phosphate-dehydrogenase).
La figure 4 représente l'inhibition de la reconnaissance de AM radiomarqué avec I125 par l'anticorps anti-AM. Des cellules glioblastomales (U87) sont incubées pendant 120 min à 25°C en présence de l125-AM (60 μci μg"1; 7 x 104 cpm par test) et des concentrations croissantes de l'anticorps. Chaque point est le résultat de trois expériences.FIG. 4 represents the inhibition of the recognition of AM radiolabelled with I 125 by the anti-AM antibody. Glioblastomal cells (U87) are incubated for 120 min at 25 ° C in the presence of 125 -AM (60 μci μg "1 ; 7 x 10 4 cpm per test) and increasing concentrations of the antibody. Each point is the result of three experiments.
La figure 5 représente les effets de AM et de l'anticorps anti-AM sur la prolifération des cellules U87 (Fig. 5A) et U373 (Fig. 5B) in vitro. Les cellules tumorales sont ensemencées à une densité de 6 x 103 cellules par puit dans des plaques 12 puits en présence de milieu MEM additionné de 2 % de FBS, 2 mM de glutamine et d'antibiotiques. On ajoute AM à une concentration de 2 x 10"7 M et l'anticorps anti-AM " AMAb2 " (12 μg /ml) en présence ou non de AM (10 μM). Sur la figure 5, C représente les cellules non traitées. Les contrôles sont constitués de " AMAb2 " dénaturé par la chaleur et de IgG (Ed.1) non relevant. Pour chaque traitement, six points sont employés pour la mesure MTT et six points sont employés pour le comptage des cellules dans un compteur Coulter. Les barres verticales représentent SEM. ** : p < 0,007 ; *** : p < 0,0001.FIG. 5 represents the effects of AM and of the anti-AM antibody on the proliferation of cells U87 (Fig. 5A) and U373 (Fig. 5B) in vitro. The tumor cells are seeded at a density of 6 × 10 3 cells per well in 12-well plates in the presence of MEM medium supplemented with 2% FBS, 2 mM glutamine and antibiotics. AM is added at a concentration of 2 x 10 "7 M and the anti-AM antibody" AMAb2 "(12 μg / ml) in the presence or not of AM (10 μM). In FIG. 5, C represents the non The controls consist of heat denatured "AMAb2" and non-relevant IgG (Ed.1). For each treatment, six points are used for MTT measurement and six points are used for counting cells in a counter. Coulter. Vertical bars represent SEM. **: p <0.007; ***: p <0.0001.
La figure 6 représente le traitement avec l'anticorps anti-adrénomédulline de xenografts de tumeurs humaines établies. Des souris porteuses de tumeurs U87, de taille approximative de 1350 +/- 100 mm3, reçoivent deux fois par semaine un traitement intra tumoral de 200 μg de AMAb2 (n=20) ou un contrôle d'IgG non relevant (n=7). Les souris non traitées avec AMAb2 sont sacrifiées entre 21 et 24 jours, en raison de la grosseur des tumeurs. Le volume moyen des tumeurs pendant une période de plus de 70 jours est montré sur la figure 6. Les grosseurs des tumeurs dans le groupe traité au PBS ne sont pas différentes de celles obtenues dans le groupe traité avec l'IgG contrôle non relevant. Culture cellulaire et tests de prolifération cellulaire.Figure 6 shows treatment with xenografts anti-adrenomedullin antibody to established human tumors. Mice with U87 tumors, approximately 1350 +/- 100 mm 3 , receive twice a week an intra-tumor treatment of 200 μg of AMAb2 (n = 20) or a non-relevant IgG control (n = 7 ). Mice not treated with AMAb2 are sacrificed between 21 and 24 days, due to the size of the tumors. The average tumor volume over a period of more than 70 days is shown in Figure 6. The sizes of the tumors in the PBS group were not different from those obtained in the non-control IgG group. Cell culture and cell proliferation tests.
Les lignées cellulaires de glioblastome humain sont obtenues de l'American Type Culture Collection (ATCC, Rockville, MD, USA) et sont cultivées en milieu essentiel minimal (MEM) (U373, U138, U87) ou dans du milieu L15 (SW1783 et SW1088), disponibles auprès de la société Life Technologies, Paris, France, en présence de pénicilline (50 U / ml), de streptomycine (50 μg / ml), de glutamine (1 mg / ml) et de sérum de veau fœtal (10 %). Les cellules sont cultivées dans une atmosphère humide 95% air / 5 % C02. Le milieu de culture est changé tous les deux jours. L'activité d'amidation et les ARNs sont préparés lorsque les cellules sont en phase exponentielle de croissance. Les effets de CGRP8^7, AM22.52, AM-|.52 et de l'anticorps de lapin anti-AM sur la prolifération cellulaire sont examinés aux temps indiqués par le test MÎT bromure de 3-(4,5-diméthyl-thiazol-2-yl)-2,5-diphényl tétrazolium (Cancer Res. 49: 4435-4440, 1989) (Cancer Res. 47 : 943-946, 1987). Les résultats sont exprimés en pourcentages des rapports T/C, où T est la densité optique (OD570) des cultures traitées (milieu MEM et CGRP8.37, AM22^2, AM^ ou anticorps de lapin anti-AM) et C est la densité optique (OD570) des cultures non traitées (milieu MEM seul).Human glioblastoma cell lines are obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and are cultured in minimal essential medium (MEM) (U373, U138, U87) or in L15 medium (SW1783 and SW1088 ), available from Life Technologies, Paris, France, in the presence of penicillin (50 U / ml), streptomycin (50 μg / ml), glutamine (1 mg / ml) and fetal calf serum (10 %). The cells are cultured in a humid atmosphere 95% air / 5% C0 2 . The culture medium is changed every two days. The amidation activity and the RNAs are prepared when the cells are in the exponential growth phase. The effects of CGRP 8 ^ 7 , AM 22 . 52 , AM- |. 52 and anti-AM rabbit antibody on cell proliferation are examined at the times indicated by the MIT test of 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide (Cancer Res. 49: 4435-4440, 1989) (Cancer Res. 47: 943-946, 1987). The results are expressed as percentages of ratios T / C, where T is the optical density (OD 570) of treated cultures (MEM medium and CGRP 8. 37, AM 22 ^ 2, AM ^ or antibodies of anti-AM rabbit) and C is the optical density (OD 570 ) of the untreated cultures (MEM medium only).
Patients et préparation des tissus.Patients and tissue preparation.
Des tissus tumoraux de patients opérés pour des gliomes sont étudiés. 5 gliomes de degré II (degré faible), 7 oligodendrogliomes anaplasiques et 13 glioblastomes (degré IV) (selon la classification histopathologique WHO) sont utilisés. Les échantillons de tumeurs sont recueillis au moment de l'intervention chirurgicale et conservés immédiatement dans l'azote liquide jusqu'à extraction des ARNs. Un tissu télencéphalique normal est prélevé sur un patient opéré dans le cadre d'un traitement contre l'épilepsie. Tous les protocoles de prélèvement de tissus ont été réalisés selon les exigences des comités institutionnels et avec l'accord des patients.Tumor tissues from patients operated on for gliomas are studied. 5 grade II gliomas (low grade), 7 anaplastic oligodendrogliomas and 13 glioblastomas (grade IV) (according to the WHO histopathological classification) are used. Tumor samples are collected at the time of surgery and immediately stored in liquid nitrogen until the RNAs are extracted. Normal telencephalic tissue is removed from a patient operated on for epilepsy treatment. All tissue sampling protocols were performed according to the requirements of the institutional committees and with the agreement of the patients.
Analyse par Northern Blot.Analysis by Northern Blot.
L'extraction des ARNs totaux est réalisée à partir des gliomes humains, du tissu télencéphalique normal et des lignées cellulaires en utilisant la méthode à l'isothiocyanate de guanidinium / phénol / chloroforme (Anal. Biochem., 162 : 156- 159, 1987) avant analyse par Northern Blot.The extraction of total RNAs is carried out from human gliomas, normal telencephalic tissue and cell lines using the method guanidinium isothiocyanate / phenol / chloroform (Anal. Biochem., 162: 156-159, 1987) before analysis by Northern Blot.
Préparation des extraits de tissus et test d'amidation.Preparation of tissue extracts and amidation test.
Les cellules sont décollées et recueillies en tampon PBS froid, et préparées pour un test d'amidation.The cells are detached and collected in cold PBS buffer, and prepared for an amidation test.
Extraction des peptides et radioimmunoessai.Peptide extraction and radioimmunoassay.
Pour mesurer l'immunomarquage anti-AM (IR-AM), les cellules sont préparées selon la procédure décrite dans FEBS Lett., 352 : 105-108, 1994. La caractérisation chromatographique de IR-AM dans le milieu de culture est réalisée par chromatographie liquide à haute performance en phase inverse, en utilisant une colonne μBondapak C18 (3,9 x 300 mm; Waters). Le milieu conditionné (15 ml) est extrait en utilisant des cartouches Sep-Pak C18. L'extrait est reconstitué avec une solution contenant 0,1 % (vol / vol) d'acide trifluoroacétique et chargé sur la colonne. L'analyse est réalisée avec un gradient linéaire d'acétonitrile contenant 0,1 % (vol / vol) d'acide trifluoroacétique, de 10 à 60 % à une vitesse de 1 ml / min / fraction pendant 50 min. Chaque fraction (1 ml) est recueillie, séchée et analysée. RT-PCR quantitative.To measure anti-AM immunostaining (IR-AM), the cells are prepared according to the procedure described in FEBS Lett., 352: 105-108, 1994. The chromatographic characterization of IR-AM in the culture medium is carried out by High performance liquid chromatography in reverse phase, using a μBondapak C18 column (3.9 x 300 mm; Waters). The conditioned medium (15 ml) is extracted using Sep-Pak C18 cartridges. The extract is reconstituted with a solution containing 0.1% (vol / vol) of trifluoroacetic acid and loaded on the column. The analysis is carried out with a linear gradient of acetonitrile containing 0.1% (vol / vol) of trifluoroacetic acid, from 10 to 60% at a speed of 1 ml / min / fraction for 50 min. Each fraction (1 ml) is collected, dried and analyzed. Quantitative RT-PCR.
Une méthode de RT-PCR (réaction de polymérase en chaîne) quantitative en temps réel est utilisée pour mesurer les variations du nombre de copies de AM ou GAPDH. L'ARN total (2 μg) libre d'ADN est transcrit en ADN complémentaire (ADNc) en présence d'un μg d'hexamères (Pharmacia Biotech, Orsay, France) et de la transcriptase inverse MMLV (de l'anglais "Moloney leukemia virus"), tel que décrit par Life Technologies Inc., Paris, France. De l'ARNm d'AM et de GAPDH humaine est amplifié (AM : amorce sens 5'-TGCCCAGACCCTTATTCGG-3' et amorce antisens 5'-AGTTGTTCATGCTCTGGCGG-3'; GAPDH : amorce sens 5'- CAAATTCCATGGCACCGTC-3' et amorce antisens 5'-A real-time quantitative RT-PCR (polymerase chain reaction) method is used to measure variations in the copy number of AM or GAPDH. Total DNA free RNA (2 μg) is transcribed into complementary DNA (cDNA) in the presence of a μg of hexamers (Pharmacia Biotech, Orsay, France) and of reverse transcriptase MMLV (from the English "Moloney leukemia virus "), as described by Life Technologies Inc., Paris, France. AM and human GAPDH mRNA is amplified (AM: primer sense 5'-TGCCCAGACCCTTATTCGG-3 'and antisense primer 5'-AGTTGTTCATGCTCTGGCGG-3'; GAPDH: primer sense 5'- CAAATTCCATGGCACCGTC-3 'and antisense primer 5'-
CCCATCTGATTTTGGAGGGA-3'), et quantifié en temps réel en utilisant le système de détection ABI Prism 7700 (PE Applied Biosystems, Foster City, CA).CCCATCTGATTTTGGAGGGA-3 '), and quantified in real time using the ABI Prism 7700 detection system (PE Applied Biosystems, Foster City, CA).
Les sondes Taq Man pour AM et GAPDH sont 5'- ACATGAAGGGTGCCTCTCGAAGCCC-3' et 5'-The Taq Man probes for AM and GAPDH are 5'- ACATGAAGGGTGCCTCTCGAAGCCC-3 'and 5'-
CCCATCACCATCTTCCAGGAGCGAG-3' respectivement. Le mélange d'amplification contient l'ADNc produit à partir de 50-150 ng de l'ARN total, 0,2 μM d'amorce, et 0,1 μM de sonde Taq Man dans 50 m de NaCI et 5 mM de Mg2+. Une PCR en deux étapes est réalisée à 35 cycles. La dénaturation est faite à 94°C pendant 20 sec, et la fixation des amorces sur la matrice / extension à 60°C pendant 30 sec. La réaction produit un produit PCR de 115 paires de base (bp) pour AM et de 101 bp pour GAPDH. Pour quantifier les résultats, des niveaux d'ARNm de AM sont normalisés aux niveaux d'ARNm de GAPDH dans la même réaction. Pour créer des courbes standard pour chaque gène, les ARNs sont produits par transcription in vitro à partir des matrices ADNc linéarisés de AM et de GAPDH par les polymérases T7 ou T3 puis rétrotranscrits en ADNc. En utilisant des sondes AM et GAPDH marquées par des fluorochromes dans les conditions expérimentales définies ci-dessus, une relation linéaire entre la concentration en ARN rétrotranscrit en ADNc et le signal de fluorescence (ΔRQ) des ARNs de AM et GAPDH dans 1- 250 pg d'ADN cible est obtenue. Pour chaque échantillon inconnu, les valeurs ΔRQ sont déterminées pour les deux gènes, et les résultats sont exprimés en fg de AM par pg de GAPDH.CCCATCACCATCTTCCAGGAGCGAG-3 'respectively. The amplification mixture contains the cDNA produced from 50-150 ng of total RNA, 0.2 μM of primer, and 0.1 μM of Taq Man probe in 50 m of NaCl and 5 mM of Mg 2+ . A two-step PCR is performed at 35 cycles. The denaturation is done at 94 ° C for 20 sec, and the fixing of the primers on the matrix / extension at 60 ° C for 30 sec. The reaction produces a PCR product of 115 base pairs (bp) for AM and 101 bp for GAPDH. To quantify the results, AM mRNA levels are normalized to GAPDH mRNA levels in the same reaction. To create standard curves for each gene, the RNAs are produced by in vitro transcription from the linearized cDNA matrices of AM and GAPDH by the T7 or T3 polymerases then retrotranscribed into cDNA. Using AM and GAPDH probes labeled with fluorochromes under the experimental conditions defined above, a linear relationship between the concentration of RNA retrotranscribed into cDNA and the fluorescence signal (ΔRQ) of the RNAs of AM and GAPDH in 1- 250 pg target DNA is obtained. For each unknown sample, the ΔRQ values are determined for the two genes, and the results are expressed in fg of AM per pg of GAPDH.
Analyse par RT-PCR des ARNm de CRLR et RAMP.RT-PCR analysis of CRLR and RAMP mRNAs.
Des amorces telles que définies dans le tableau 1 figurant en annexe de la présente demande de brevet sont sélectionnées pour être spécifiques de CRLR, RAMP2 et RAMP3. Les réactions de PCR initiées par les jeux d'amorces spécifiques pour CRLR, RAMP2 et RAMP3 sont réalisées avec de l'ADNc produit à partir de lignées cellulaires de gliome et d'échantillon prélevé sur un gliome humain. Les paramètres de cycles sont les suivants : Etape de dénaturation initiale : 94°C, 5 min,Primers as defined in Table 1 appended to this patent application are selected to be specific for CRLR, RAMP2 and RAMP3. The PCR reactions initiated by the specific primer sets for CRLR, RAMP2 and RAMP3 are carried out with cDNA produced from glioma cell lines and from a sample taken from a human glioma. The cycle parameters are as follows: Initial denaturation stage: 94 ° C., 5 min,
35 cycles : fixation des amorces sur la matrice : 40 sec. puis extension I polymérisation à 68°C, 50 sec, puis dénaturation 94°C, 30 sec, la dernière extension étant prolongée de 10 min. Les températures de fixation des amorces sur la matrice dépendent du couple d'amorces utilisé et sont indiquées dans le tableau 1. Les produits de PCR sont séparés par électrophorèse sur un gel d'agarose à 1 % dans un tampon Tris / borate contenant 0,5 μg / ml de bromure d'éthidium; les gels sont rincés en tampon NaCI 1 ,5 M, NaOH 0,5 N pendant 15 min et ensuite pendant 30 min dans Tris-HCI 1 M, pH 8,0, NaCI 1 ,5 M avant transfert sur une membrane Hybond-N. Les filtres sont mis à hybrider avec les sondes indiquées dans le tableau 1 , lavés et mis en autoradiographie. Production des anticorps anti-AM.35 cycles: fixing of the primers on the matrix: 40 sec. then extension I polymerization at 68 ° C, 50 sec, then denaturation 94 ° C, 30 sec, the last extension being extended by 10 min. The temperatures for fixing the primers to the matrix depend on the pair of primers used and are indicated in Table 1. The PCR products are separated by electrophoresis on a 1% agarose gel in a Tris / borate buffer containing 0, 5 μg / ml ethidium bromide; the gels are rinsed in 1.5 M NaCI buffer, 0.5 N NaOH for 15 min and then for 30 min in 1 M Tris-HCI, pH 8.0, 1.5 M NaCI before transfer to a Hybond-N membrane . The filters are hybridized with the probes indicated in Table 1, washed and put into autoradiography. Production of anti-AM antibodies.
Des lapins néo-zélandais femelles sont immunisés trois fois en sous-cutané avec de l'AM humaine (Bachem) (120 μg AM équivalent par injection : la première dose dans un adjuvant de Freund complet, les deux dernières dans un adjuvant de Freund incomplet). Les sérums sont sélectionnés pour leur activité anti-AM, puis purifiés par affinité sur colonne de protéine A Sépharose (Amersham Pharmacia Biotech).New Zealand female rabbits are immunized three times subcutaneously with human AM (Bachem) (120 μg AM equivalent per injection: the first dose in a complete Freund's adjuvant, the last two in an incomplete Freund's adjuvant ). The sera are selected for their anti-AM activity, then affinity purified on a protein A Sepharose column (Amersham Pharmacia Biotech).
Reconnaissance de AM marqué à I125.Recognition of AM marked at I 125 .
Des cellules de glioblastome sont cultivées dans des plaques 24 puits jusqu'à confluence (1 x 105 cellules / puit) et déprivées en sérum pendant 24 h après lavage avec un tampon PBS sans Ca + additionné de 0,2 % de sérum albumine bovine (BSA) et de Bacitracine (100 mg / ml, Sigma), les cellules sont incubées à 25°C pendant 120 min avec le marqueur radioactif (I125) en présence ou en absence d'un excès (10"6 M) de AM non radiomarquée. Le marquage à l'iode de AM humaine (Amh) synthétique est réalisée par la méthode à la chloranine T et le produit est purifié par chromatographie liquide à haute performance en phase inverse. De l'Amh monoiodée (I125) (SA, 350 Ci / mmol) est utilisée dans les expériences. Dans les études d'inhibition de reconnaissance (de l'anglais "binding"), les cellules sont incubées avec le marqueur radiomarqué et des concentrations croissantes d'anticorps anti-AM. A la fin de la période d'incubation, les cellules sont lavées avec du PBS froid contenant 0,2 % de BSA, solubilisées avec de l'hydroxyde de sodium 0,2 M, et la radioactivité retenue est mesurée par un spectromètre-γ. La fixation spécifique est obtenue par soustraction de la mesure de la reconnaissance non spécifique obtenue en présence d'AMh non marquée en excès de la mesure de la reconnaissance totale. Les données représentent la moyenne de trois expériences, chacune réalisée trois fois. Animaux.Glioblastoma cells are cultured in 24-well plates until confluence (1 × 10 5 cells / well) and deprived in serum for 24 h after washing with PBS buffer without Ca + added with 0.2% bovine serum albumin (BSA) and Bacitracin (100 mg / ml, Sigma), the cells are incubated at 25 ° C for 120 min with the radioactive marker (I 125 ) in the presence or absence of an excess (10 "6 M) of Non-radiolabelled AM The synthetic iodine labeling of human AM (Amh) is carried out by the chloranine T method and the product is purified by reverse phase high performance liquid chromatography. Monoiodized Amh (I 125 ) (SA, 350 Ci / mmol) is used in the experiments. In the recognition binding inhibition studies, the cells are incubated with the radiolabeled marker and increasing concentrations of anti-AM antibodies. At the end of the incubation period, the cells are washed with Cold PBS containing 0.2% BSA, solubilized with 0.2 M sodium hydroxide, and the radioactivity retained is measured by a γ spectrometer. The specific binding is obtained by subtracting the measurement of non-specific recognition obtained in the presence of unmarked AMh in excess of the measurement of total recognition. The data represents the average of three experiments, each performed three times. Animals.
Des souris NMRI mâles (Nu / Nu) athymiques de 4 à 5 semaines (Janvier, Laval Le Genest, France), sont placées dans des cages stériles sous flux laminaire fermé, en température contrôlée et avec des périodes de 12 h de lumière et 12 h d'obscurité, et alimentées et désaltérées ad libitum. Thérapie anti-tumorale in vivo.Athymic male NMRI mice (Nu / Nu) from 4 to 5 weeks (Janvier, Laval Le Genest, France), are placed in sterile cages under closed laminar flow, at controlled temperature and with periods of 12 h of light and 12 h of darkness, and fed and quenched ad libitum. In vivo anti-tumor therapy.
Des cellules U87 (3 x 106 cellules / souris) en phase exponentielle de croissance sont injectées en sous-cutané dans les flancs des souris athymiques. Après deux semaines, les tumeurs ont grossi jusqu'à un volume d'environ 1350 +/- 170 mm3. Le traitement est initié après 14 jours. L'anticorps anti-AM AMAb2 (200 μg d'IgG purifié) est injecté dans la tumeur (20 souris) dans un volume de 0,2 ml, deux fois par semaine. Un contrôle négatif, un anti-lgG de lapin dirigé contre le peptide dérivé du précurseur de Pendothéline de rat ou le tampon phosphate (PBS) seul sont utilisés. Chaque groupe contrôle comporte sept souris. Chaque souris dans les groupes traités par immunoglobuline reçoit un total de 400 μg d'immunoglobuline par semaine pendant la durée du traitement. Les tumeurs sont mesurées deux fois par semaine pendant le traitement. Le volume des tumeurs est calculé selon la formule volume = longueur x largeur x hauteur x 0,5236 (pour une forme elliptique). Les données sont exprimées en moyenne +/- SEM. Les analyses statistiques sont réalisées en utilisant la méthode ANOVA suivie par le test de différence significative de Fisher (Statview 512, Brain Power Inc., Calabasas, CA, USA). Une valeur de p < 0,05 est considérée comme significative.U87 cells (3 x 10 6 cells / mouse) in exponential growth phase are injected subcutaneously into the flanks of athymic mice. After two weeks, the tumors have grown to a volume of approximately 1350 +/- 170 mm 3 . The treatment is started after 14 days. The anti-AM AMAb2 antibody (200 μg of purified IgG) is injected into the tumor (20 mice) in a volume of 0.2 ml, twice a week. A negative control, a rabbit anti-IgG directed against the peptide derived from the rat endothelin precursor or the phosphate buffer (PBS) alone are used. Each control group has seven mice. Each mouse within groups treated with immunoglobulin receives a total of 400 μg of immunoglobulin per week for the duration of treatment. Tumors are measured twice a week during treatment. The volume of the tumors is calculated according to the formula volume = length x width x height x 0.5236 (for an elliptical shape). Data are expressed as an average +/- SEM. Statistical analyzes are performed using the ANOVA method followed by Fisher's significant difference test (Statview 512, Brain Power Inc., Calabasas, CA, USA). A value of p <0.05 is considered significant.
Expression d'ARNm de PAM dans les cellules gliomales humaines. L'ARN total de gliome humain, de lignées cellulaires de gliome humain et de tissu de cerveau normal est préparé pour analyser le niveau d'expression de PAM. Avec une sonde de 2,2 kilobases (kb), le transcript correspondant à la PAM est mis en évidence par Northern Blot à 4 kb. Un fort niveau d'expression est mis en évidence dans le gliome humain (Fig. 1A) et les lignées cellulaires (Fig. 1 B) en comparaison des tissus normaux. Après décrochage des membranes, un second marquage est réalisé avec la sonde ADNc préparée à partir d'ARNr 18S. Dans les gliomes, le niveau d'expression de PAM, quantifié et normalisé par rapport à l'ARN ribosomal 18S, apparaît être corrélé à l'agressivité de la tumeur. Les niveaux d'ARNm PAM sont 9 à 15 fois plus élevés dans les glioblastomes malins et 4 à 6 fois plus élevés dans les gliomes de faible degré que dans les tissus de cerveau normal. La majorité des lignées cellulaires dérivées de glioblastomes humains expriment de façon constante de hauts niveaux d'ARNm de PAM (Fig. 1 B). Activité PAM dans les lignées de cellules. L'activité d'amidation des extraits de chaque lignée cellulaire est testée en utilisant un substrat α-N-acetyl-Tyr-Val-Gly à pH 5,5. Les lignées cellulaires U373 et SW1783 gliome de haut degré présentent des niveaux d'activité PAM plus élevés (77 +/- 4 pmol / mg de protéine /h) que les lignées U87, U 138 et SW1088 (55 +/- 4,3 pmol / mg de protéine / h). L'expression de PAM à la fois dans les tumeurs et les lignées gliomales démontre leur capacité à produire des peptides α-amidés. Expression de l'ARNm de AM dans les lignées cellulaires.Expression of PAM mRNA in human gliomal cells. Total RNA from human glioma, human glioma cell lines and normal brain tissue is prepared to analyze the level of PAM expression. With a 2.2 kilobase (kb) probe, the transcript corresponding to PAM is highlighted by Northern Blot at 4 kb. A high level of expression is demonstrated in the human glioma (Fig. 1A) and the cell lines (Fig. 1B) in comparison with normal tissues. After detaching the membranes, a second labeling is carried out with the cDNA probe prepared from 18S rRNA. In gliomas, the level of PAM expression, quantified and normalized with respect to 18S ribosomal RNA, appears to be correlated with the aggressiveness of the tumor. PAM mRNA levels are 9 to 15 times higher in malignant glioblastomas and 4 to 6 times higher in low-grade gliomas than in normal brain tissue. The majority of cell lines derived from human glioblastomas consistently express high levels of PAM mRNA (Fig. 1 B). PAM activity in cell lines. The amidation activity of the extracts from each cell line is tested using an α-N-acetyl-Tyr-Val-Gly substrate at pH 5.5. U373 and SW1783 high-grade glioma cell lines have higher PAM activity levels (77 +/- 4 pmol / mg protein / h) than U87, U 138 and SW1088 lines (55 +/- 4.3 pmol / mg protein / h). The expression of PAM in both tumors and gliomal lines demonstrates their ability to produce α-amide peptides. Expression of AM mRNA in cell lines.
L'expression de différents peptides α-amidés connus pour avoir des propriétés mitogènes sur les cellules tumorales est analysée par RT-PCR dans différentes lignées de gliomes. Parmi tous les peptides analysés, AM présente un niveau d'expression très élevé. Le niveau d'expression de l'ARNm AM est analysé par Northern Blot sur des lignées gliomales et des tissus normaux humains (Biochem. Biophys. Res. Commun., 194 : 720-725, 1993). La taille du transcript est d'environ 1 ,6 kb. La quantité d'ARNm de AM est ensuite normalisée par rapport à la quantité d'ARN ribosomal 18S (Fig. 2). Toutes les lignées cellulaires gliomales testées expriment l'ARNm de AM, tandis qu'aucune expression n'est détectée dans les tissus non tumoraux.The expression of different α-amide peptides known to have mitogenic properties on tumor cells is analyzed by RT-PCR in different glioma lines. Among all the peptides analyzed, AM has a very high level of expression. The level of expression of the AM mRNA is analyzed by Northern Blot on gliomal lines and normal human tissues (Biochem. Biophys. Res. Commun., 194: 720-725, 1993). The size of the transcript is approximately 1.6 kb. The amount of AM mRNA is then normalized with respect to the amount of 18S ribosomal RNA (Fig. 2). All the gliomal cell lines tested express the AM mRNA, while no expression is detected in non-tumor tissues.
Production et sécrétion de AM dans les lignées cellulaires.Production and secretion of AM in cell lines.
L'immunomarquage anti-AM est positif dans les extraits cellulaires et le milieu de culture. Toutes les lignées gliomales produisent donc et sécrètent de l'AM. La quantité d'AM obtenue est indiquée dans le tableau 2 figurant en annexe de la présente demande de brevet.Anti-AM immunostaining is positive in cell extracts and the culture medium. All gliomal lines therefore produce and secrete AM. The quantity of AM obtained is indicated in Table 2 in the appendix to this patent application.
Expression de l'ARNm de AM dans le gliome humain.Expression of AM mRNA in human glioma.
De l'ARN total est préparé à partir de prélèvements de tumeurs de 25 gliomes comprenant 13 gliomes de haut degré (IV), 5 gliomes de bas degré (II) et 7 oligodendrogliomes anaplasiques. Les niveaux d'expression de l'ARNm de AM sont indiqués sur la figure 3. La quantification des transcripts d'ARNm de AM révèle un plus fort niveau d'expression de l'ARNm de AM dans les glioblastomes, comparés aux oligodendrogliomes de bas degré ou anaplasiques.Total RNA is prepared from samples of 25 glioma tumors comprising 13 high degree (IV) gliomas, 5 low degree (II) gliomas and 7 anaplastic oligodendrogliomas. The levels of expression of AM mRNA are indicated in FIG. 3. The quantification of AM mRNA transcripts reveals a higher level of expression of AM mRNA in glioblastomas, compared to the oligodendrogliomas of low degree or anaplastic.
Expression des ARNm de RAMP et CRLR dans les lignées cellulaires.Expression of RAMP and CRLR mRNAs in cell lines.
Pour déterminer si les gliomes humains et les lignées cellulaires expriment CRLR, RAMP2 et RAMP3, une analyse par Southern Blot des produits de RT-PCR est réalisée. L'analyse des produits avec les sondes correspondantes révèle des bandes aux tailles attendues (tableau 1) correspondant à l'ARNm codant pour CRLR ainsi que RAMP2 et RAMP3. Aucune bande n'apparaît dans les contrôles sans transcriptase inverse. Caractérisation de l'anticorps anti-AM.To determine whether human gliomas and cell lines express CRLR, RAMP2 and RAMP3, a Southern Blot analysis of the RT-PCR products is carried out. Analysis of the products with the corresponding probes reveals bands of the expected sizes (Table 1) corresponding to the mRNA coding for CRLR as well as RAMP2 and RAMP3. No band appears in the controls without reverse transcriptase. Characterization of the anti-AM antibody.
L'anticorps anti-AM polyclonal (sérum ou IgG purifié) reconnaît bien AM pleine taille mais ne montre aucune réactivité croisée avec des peptides apparentés à AM, tel que cela apparaît sur le tableau 3 figurant en annexe de la présente demande de brevet. La calcitonine, CGRPι.3 ) CGRP8^7 et l'amyline ne montrent qu'une fixation non significative avec l'anticorps. De plus, aucune réaction croisée avec les peptides ACTH (humaine 1-24), endotheline-1 (humaine), AVP (humaine), CRF (humaine), TRH, substance P, ANF (humaine) n'est observée.The polyclonal anti-AM antibody (serum or purified IgG) clearly recognizes full-size AM but does not show any cross-reactivity with peptides related to AM, as it appears in Table 3 appearing in the appendix to the present patent application. Calcitonin, CGRPι. 3) CGRP 8 ^ 7 and the amylin only show a non-significant fixation with the antibody. In addition, no cross-reaction with the peptides ACTH (human 1-24), endotheline-1 (human), AVP (human), CRF (human), TRH, substance P, ANF (human) is observed.
La capacité de l'anticorps à bloquer la fixation de AM-I125 sur son récepteur dans des cellules U87 est testée (Fig. 4). L'anticorps bloque l'interaction récepteur - AM de façon dose dépendante. Effets de AM et de l'anticorps anti-AM sur la prolifération des cellules U87 et U373 in vitro.The ability of the antibody to block the binding of AM-I 125 to its receptor in U87 cells is tested (Fig. 4). The antibody blocks the receptor-AM interaction in a dose-dependent manner. Effects of AM and anti-AM antibody on the proliferation of U87 and U373 cells in vitro.
Des cellules U87 et U373 sont cultivées en présence de AM 2 x 10"7 M, et les effets du peptide sur la prolifération cellulaire sont examinés par une méthode MTT. Les figures 5A et 5B montrent que AM, à une concentration de 2 x 10"7 M, stimule la prolifération de cellules U87 de 15 % (p < 0,02) et U373 de 20 % (pU87 and U373 cells are cultured in the presence of AM 2 x 10 "7 M, and the effects of the peptide on cell proliferation are examined by an MTT method. 5A and 5B show that AM at a concentration of 2 x 10 "7 M, stimulates the proliferation of U87 cells by 15% (p <0.02) and U373 by 20% (p
<0,001) après 8 jours de traitement.<0.001) after 8 days of treatment.
Un anticorps anti-AM polyclonal (purifié IgG) est testé pour son effet inhibiteur sur la prolifération des cellules U87 et U373 cultivées en présence d'anticorps. Après 7 jours de traitement, l'anticorps à une concentration de 12 μg / ml inhibe la prolifération de U87 et U373 de 33 % (p < 0,002) et de 50 % (p < 0,001) respectivement (Fig. 5A et 5B).A polyclonal anti-AM antibody (purified IgG) is tested for its inhibitory effect on the proliferation of U87 and U373 cells cultured in the presence of antibodies. After 7 days of treatment, the antibody at a concentration of 12 μg / ml inhibits the proliferation of U87 and U373 by 33% (p <0.002) and 50% (p <0.001) respectively (Fig. 5A and 5B).
Effets de l'anticorps anti-AM in vivo sur des souris présentant des tumeurs. La capacité de l'anticorps anti-AM à inhiber la croissance in vivo a été testée sur des cellules U87 xénogreffées sur souris. Six jours après inoculation dans les souris, la lignée cellulaire produit une masse tumorale palpable (506 +/- 70 mm3). Au 14è e jour (1 350 +/- 170 mm3), les tumeurs sont soumises à trois traitements : tampon de PBS, IgG de spécificité non relevante, anticorps anti-AM (IgG purifié).Effects of the anti-AM antibody in vivo on mice with tumors. The ability of the anti-AM antibody to inhibit growth in vivo was tested on U87 cells xenografted in mice. Six days after inoculation in the mice, the cell line produces a palpable tumor mass (506 +/- 70 mm 3 ). 14th th day (1350 +/- 170 mm 3) tumors are subjected to three treatments: PBS buffer, non relevante specificity of IgG anti-AM antibody (purified IgG).
La figure 6 montre les courbes de croissance de tumeurs de souris porteuses d'un glioblastome humain U87, qui sont traités tous les trois jours avec l'anticorps anti-AM, ou le contrôle IgG non relevant. Le traitement est administré par injection intra-tumorale et la croissance est déterminée en fonction du volume tumoral au cours du temps. Après 21 jours de traitement avec l'anticorps à une dose de 200 μg deux fois par semaine, le volume moyen de la tumeur est réduit à 646 +/-FIG. 6 shows the growth curves of tumors of mice carrying a human glioblastoma U87, which are treated every three days with the anti-AM antibody, or the non-relevant IgG control. The treatment is administered by intra-tumor injection and the growth is determined as a function of the tumor volume over time. After 21 days of treatment with the antibody at a dose of 200 μg twice a week, the average volume of the tumor is reduced to 646 +/-
166 mm3 (p < 0,001) par comparaison avec le groupe de contrôle qui atteint un volume de tumeur de 2 150 +/- 160 mm3. Des injections de l'anticorps pendant 7 semaines conduisent à une très forte diminution de la croissance tumorale, une régression de la tumeur (masse tumorale inférieure à 40 mm3 chez toutes les souris) ainsi qu'une augmentation significative du nombre de survivants. Le volume tumoral moyen à la fin de l'expérience représente 2 % du volume tumoral moyen initial. La croissance des tumeurs chez les souris des groupes de contrôle a conduit au sacrifice des animaux moins de 6 semaines après l'injection des cellules. Les poids moyens des tumeurs dans les contrôles et les souris traitées avec l'anticorps étaient de 3,8 +/- 0,5 g et 1,18 +/- 0,3 g respectivement (p < 0,001) après 20 jours de traitement. La diminution du poids des tumeurs est de 68,9 % et 91,5 % après 24 et 42 jours de traitement respectivement chez les animaux traités avec l'anticorps, en comparaison avec les groupes de contrôle. 166 mm 3 (p <0.001) by comparison with the control group which reaches a tumor volume of 2,150 +/- 160 mm 3 . Injections of the antibody for 7 weeks lead to a very strong decrease in tumor growth, a regression of the tumor (tumor mass less than 40 mm 3 in all the mice) as well as a significant increase in the number of survivors. The average tumor volume at the end of the experiment represents 2% of the initial average tumor volume. The growth of the tumors in the control group mice led to the sacrifice of the animals less than 6 weeks after the injection of the cells. Average tumor weights in controls and antibody treated mice were 3.8 +/- 0.5 g and 1.18 +/- 0.3 g respectively (p <0.001) after 20 days of treatment. The decrease in tumor weight is 68.9% and 91.5% after 24 and 42 days of treatment respectively in the animals treated with the antibody, in comparison with the control groups.
ANNEXEANNEX
TABLEAU 1TABLE 1
Amorces utilisées pour la détection de l'expression de CRLR, RAMP2 et RAMP3.Primers used for the detection of the expression of CRLR, RAMP2 and RAMP3.
La référence correspond au numéro Genbank.
ANNEXE
TABLEAU 2TABLE 2
Quantité de AM dans les extraits cellulaires et le milieu de culture des lignées cellulaires gliomales.Amount of AM in cell extracts and culture medium of gliomal cell lines.
ANNEXE
TABLEAU 3TABLE 3
Réactivité de l'anticorps anti-AM avec des peptides apparentés.Reactivity of anti-AM antibody with related peptides.
Claims
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2002238673A AU2002238673A1 (en) | 2001-02-20 | 2002-02-20 | Human adrenomedullin-specific antibody, pharmaceutical composition containing same, therapeutic uses thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR01/02291 | 2001-02-20 | ||
| FR0102291A FR2821080B1 (en) | 2001-02-20 | 2001-02-20 | SPECIFIC ANTIBODIES TO HUMAN ADRENOMEDULLINE, PHARMACEUTICAL COMPOSITION CONTAINING SAME, ITS THERAPEUTIC APPLICATIONS |
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| WO2002066492A2 true WO2002066492A2 (en) | 2002-08-29 |
| WO2002066492A3 WO2002066492A3 (en) | 2003-02-27 |
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| PCT/FR2002/000638 WO2002066492A2 (en) | 2001-02-20 | 2002-02-20 | Human adrenomedullin-specific antibody, pharmaceutical composition containing same, therapeutic uses thereof |
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| AU (1) | AU2002238673A1 (en) |
| FR (1) | FR2821080B1 (en) |
| WO (1) | WO2002066492A2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2964103A1 (en) * | 2010-08-30 | 2012-03-02 | Univ Provence Aix Marseille 1 | ANTIBODIES BINDING TO ADRENOMEDULLINE AND RECEPTORS OF ADRENOMEDULLINE AND THEIR USES AS A MEDICINAL PRODUCT |
| EP2589963A1 (en) * | 2011-11-04 | 2013-05-08 | Université de Provence (Aix-Marseille 1) | Diagnostic and treatment of an androgen-independent prostate cancer |
| US9862771B2 (en) | 2008-12-23 | 2018-01-09 | Amgen Inc. | Human CGRP receptor binding proteins |
| US10259877B2 (en) | 2015-04-24 | 2019-04-16 | Amgen Inc. | Methods for treating or preventing migraine headache |
| US10934362B2 (en) | 2014-09-15 | 2021-03-02 | Amgen Inc. | Bi-specific anti-CGRP receptor/PAC1 receptor antigen binding proteins and uses thereof |
| US11407838B2 (en) | 2018-04-02 | 2022-08-09 | Amgen Inc. | Erenumab compositions and uses thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1575417A4 (en) * | 2002-09-30 | 2007-07-04 | Bayer Pharmaceuticals Corp | Methods for prediction and prognosis of cancer, and monitoring cancer therapy |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2774769B2 (en) * | 1993-04-26 | 1998-07-09 | 賢治 寒川 | Adrenomedullin |
| ATE180832T1 (en) * | 1995-08-18 | 1999-06-15 | Us Gov Health & Human Serv | FUNCTIONAL ROLE OF ADRENOMEDULLIN(AM) AND THE GENE RELATED PRODUCT(PAMP) IN HUMAN PATHOLOGY AND PHYSIOLOGY |
-
2001
- 2001-02-20 FR FR0102291A patent/FR2821080B1/en not_active Expired - Fee Related
-
2002
- 2002-02-20 WO PCT/FR2002/000638 patent/WO2002066492A2/en not_active Application Discontinuation
- 2002-02-20 AU AU2002238673A patent/AU2002238673A1/en not_active Abandoned
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9862771B2 (en) | 2008-12-23 | 2018-01-09 | Amgen Inc. | Human CGRP receptor binding proteins |
| US12134652B2 (en) | 2008-12-23 | 2024-11-05 | Amgen Inc. | Human CGRP receptor binding proteins |
| US12122841B2 (en) | 2008-12-23 | 2024-10-22 | Amgen Inc. | Human CGRP receptor binding proteins |
| WO2012029023A1 (en) * | 2010-08-30 | 2012-03-08 | Universite De Provence - Aix-Marseille 1 | Antibodies that bind to adrenomedullin and to adrenomedullin receptors and the uses thereof as drugs |
| US9474794B2 (en) | 2010-08-30 | 2016-10-25 | Universite D'aix-Marseille | Antibodies that bind to adrenomedullin and to adrenomedullin receptors and the uses thereof as drugs |
| FR2964103A1 (en) * | 2010-08-30 | 2012-03-02 | Univ Provence Aix Marseille 1 | ANTIBODIES BINDING TO ADRENOMEDULLINE AND RECEPTORS OF ADRENOMEDULLINE AND THEIR USES AS A MEDICINAL PRODUCT |
| EP2589963A1 (en) * | 2011-11-04 | 2013-05-08 | Université de Provence (Aix-Marseille 1) | Diagnostic and treatment of an androgen-independent prostate cancer |
| WO2013065019A1 (en) * | 2011-11-04 | 2013-05-10 | Universite D'aix Marseille | Diagnostic and treatment of an androgen-independent prostate cancer |
| US11919964B2 (en) | 2014-09-15 | 2024-03-05 | Amgen Inc. | Bi-specific anti-CGRP receptor/PAC1 receptor antigen binding proteins and uses thereof |
| US10934362B2 (en) | 2014-09-15 | 2021-03-02 | Amgen Inc. | Bi-specific anti-CGRP receptor/PAC1 receptor antigen binding proteins and uses thereof |
| US10259877B2 (en) | 2015-04-24 | 2019-04-16 | Amgen Inc. | Methods for treating or preventing migraine headache |
| US11466090B2 (en) | 2015-04-24 | 2022-10-11 | Amgen Inc. | Methods for treating or preventing migraine headache |
| US11407838B2 (en) | 2018-04-02 | 2022-08-09 | Amgen Inc. | Erenumab compositions and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2821080B1 (en) | 2003-12-19 |
| WO2002066492A3 (en) | 2003-02-27 |
| AU2002238673A1 (en) | 2002-09-04 |
| FR2821080A1 (en) | 2002-08-23 |
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