WO2003018048A1 - Utilisation de molecules solubles hla de classe 1 pour inhiber l'angiogenese - Google Patents
Utilisation de molecules solubles hla de classe 1 pour inhiber l'angiogenese Download PDFInfo
- Publication number
- WO2003018048A1 WO2003018048A1 PCT/FR2002/002812 FR0202812W WO03018048A1 WO 2003018048 A1 WO2003018048 A1 WO 2003018048A1 FR 0202812 W FR0202812 W FR 0202812W WO 03018048 A1 WO03018048 A1 WO 03018048A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hla
- cells
- molecule
- soluble
- molecules
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the field of inhibition of angiogenesis involved in numerous pathological proliferative syndromes.
- the invention relates to the use of soluble molecules of the Major Histocompatibility Complex (MHC) and more particularly to the use of soluble HLA class 1 molecules, such as HLA-Gs or HLA-B7S, for the "" preparation pharmaceutical compositions useful for inhibiting angiogenesis, and therefore intended for the treatment of angiogenic pathologies involving endothelial cells and in particular to check their tumor proliferation.
- MHC Major Histocompatibility Complex
- HLA-Gs or HLA-B7S soluble HLA class 1 molecules
- the invention also contemplates the use of these molecules to vectorize substances of therapeutic or diagnostic interest at the level of endothelial cells involved in a process of pathological angiogenesis.
- the invention also relates to the preparation of antibodies directed against these molecules and to the compositions containing them to promote angiogenesis and treat pathologies requiring the proliferation of endothelial cells.
- Endothelial cells line the inside of all of the body's blood vessels. These cells have a quiescent phenotype, they hardly proliferate, in fact only 0.01% of endothelial cells are engaged in cell division at any given time. The physiological role of endothelial cells is very vast, they participate in many vital phenomena for the organism: blood coagulation, maintenance of blood pressure, recruitment of circulating cells, synthesis of growth factors.
- HLA-G class Ib gene located on the same chromosomal region as the other genes of the major histocompatibility complex (MHC) of class la (HLA-A, -B and -C) is characterized by structural properties, some of which seem unique (Le Bouteiller and Blaschitz, 1999; Le Bouteiller et al., 1999).
- MHC major histocompatibility complex
- HLA-G Four membrane isoforms of HLA-G have been described, they are due to the alternative splicing of one or two exons coding for the external domains of the molecule (Ishitani and Geraghty, 1992; Kirszenbaum et al., 1994).
- HLA-G protein has been detected in various subpopulations of placental cell ions: the extravillous cytotrophoblast, certain macrophages present in the chorionic villi, and the endothelial cells of the fetal vessels as well as the cells of the amniotic epithelium (Blaschitz et al. , 1999; Hammer et al., 1997).
- HLA-G protein soluble HLA-G was detected "" daris' amniotic fluid as well as "in the peripheral blood of pregnant women (Fournel et al., 1999; Puppo et al., 1999; Rebmann et al., 1999; Fournel et al., 2000a)
- the presence of HLA-G in the placenta suggests that this molecule could exert a role favoring the maintenance of gestation by preventing rejection of the fetus by the mother (Le Bouteiller and Blaschitz, 1999; Le Bouteiller et al., 1999. Since the HLA-G molecule is not polymorphic, it cannot by itself induce maternal anti-paternal alloimmune reaction.
- the HLA-G molecule can recognize cells T activated, independently of the T cell Receptor, via CD8 molecules, and induce apoptosis of these cells in vitro (Fournel et al. 2000b).
- the inventors are interested in the mechanism of apoptosis induction of activated CD8 + T cells which could promote in vivo the local elimination in the decidua, of alloreactive maternal CD8 T cells.
- Soluble HLA-G present in maternal blood during gestation (Puppo et al., 1999; Rebmann et al., 1999) could exert the same effect and thus contribute to the maintenance of a state of tolerance towards HLA-G ( Fournel et al., 2000b).
- the membrane HLA-G molecule present on the cells of the extravillous cytotrophoblast, can be recognized by NK receptors of the inhibitor type (Lanier, 1998). Such recognition can either inhibit the lysis by NK decidual cells of these trophoblastic cells (Loke and King, 1995), or induce the secretion of specific cytokines promoting gestation (Maejima et al., 1997).
- the soluble HLA-G molecule could exert a modulating role in placental angiogenesis " (Le Bouteiller and Blaschitz, 1999; Le Bouteiller et al., 1999). Thus, a certain number of observations have suggested that this molecule could exert a stimulating function for maternal endothelial cells and / or for trophoblastic cells:
- HLA-G has been detected by immunohistochemistry on placenta sections in certain endothelial cells (Blaschitz et al., 1997) using certain anti-HLA-G antibodies, the placental expression of HLA-G is greatly reduced, or even absent, in pre-eclamptic placentas (Hara et al., 1996), pathology associated with a defect in vascularisation (Taylor, 1997), - the endothelial cells lining the maternal spiral arteries present in the decidua are gradually eliminated and replaced by endovascular trophoblastic cells, which themselves express HLA-G during placentation (Proll et al., 1999).
- HLA class I molecules have been described as being able to structurally associate with certain membrane receptors, including insulin receptors (Kittur et al., 1987) and EGF receptors (Schreiber et al., 1984). Stagsted et al. (1990) had also demonstrated that peptides derived from HLA class I molecules could regulate the function of insulin receptors. However, to date no description of any non-immunological role of the soluble form of HLA Gl has been reported and there are also no experimental data demonstrating the presence of HLA-Gs receptors on endothelial cells. No biological effect of HLA-Gs on these cells or in angiogenesis has been described.
- HLA-Gs soluble HLA-G molecule
- ICAM-1 lymphocyte recognition molecule
- the soluble HLA-G and HLA-B7 molecules are capable of inhibiting the tumor progression of xenografts of adenocarcinoma cells of the human prostate grafted in mice immunosuppressed nude. More particularly, the HLA-Gls molecule exerts this anti-proliferative effect on endothelial cells originating from different species (Man, beef, pig), which suggests an important function conserved in different species of mammals.
- a subject of the present invention is therefore the use of a class 1 HLA soluble molecule or of a nucleic acid molecule comprising a polynucleotide sequence coding for a HLA soluble molecule of class 1 for the preparation of pharmaceutical compositions intended to inhibit angiogenesis or to detect angiogenic sites.
- a class 1 HLA soluble molecule or of a nucleic acid molecule comprising a polynucleotide sequence coding for a HLA soluble molecule of class 1 for the preparation of pharmaceutical compositions intended to inhibit angiogenesis or to detect angiogenic sites.
- the soluble HLA class 1 molecules are capable of inhibiting or stimulating the activity of VEGF receptors.
- the invention relates more particularly to HLA-Gls or HLA-B7s molecules, and the polynucleotide sequences coding for these.
- HLA-Gs molecule is a non-polymorphic molecule, unlike HLA molecules of class la, known as classics, which, themselves, are extremely polymorphic and can therefore induce an alloreaction if they are injected into individuals not Identical HLA.
- HLA-Gs molecule make it particularly useful for its use in the context of angiogenic pathologies involving abnormalities of proliferation of endothelial cells.
- two soluble HLA-G isoforms have also been demonstrated, such isoforms constitute functionally equivalent molecules.
- the invention relates to derivatives of soluble HLA class 1 molecules having anti-angiogenic properties that a person skilled in the art is able to test using the methods reported in the experimental part below. They may be fragments, in truncated form, sequences modified by deletion, addition or deletion of one or more amino acids. Likewise, the invention relates to polynucleotide sequences coding for said derivatives.
- nucleic acid molecule comprising a polynucleotide sequence encoding a soluble HLA class 1 molecule, said sequence being placed under the control of sequences regulating its expression.
- a """ such nucleic acid molecule ""” is " a vector, such as, for example, a plasmid, a recombinant virus, a cell transfected with a plasmid or viral expression vector.
- Such a nucleic acid molecule may be used in gene therapy or cell therapy protocols consisting in administering said molecule or cells transformed by said molecule to an individual so as to express a soluble HLA class 1 molecule and thus inhibit angiogenesis.
- Soluble HLA class 1 molecules are particularly useful for treating pathologies involving endothelial cells engaged in an angiogenesis process. Thus, these molecules are particularly suitable for the preparation of drugs capable of preventing or inhibiting angiogenesis and therefore the proliferation of endothelial cells.
- the soluble HLA class 1 molecule is associated in the compositions according to the invention with any pharmaceutically acceptable vehicle known to a person skilled in the art suitable for the mode of administration used.
- the compositions according to the invention can be administered by systemic route or local injection. They include an adequate amount of active agents consisting of at least one soluble HLA class I molecule.
- angiogenesis By way of example of pathologies the treatment of which requires preventing or inhibiting angiogenesis, mention may be made of cancer, diabetic retinopathies, rheumatoid arthritis, chronic rejection of transplants such as the cornea or the kidney or acute rejection observed in xenotransplantation, angiomas, angiosarcomas, in particular Castelman's disease or Kaposi's sarcoma.
- the research carried out in the context of the present invention has made it possible to demonstrate the existence of receptors for the HLA-Gls molecule on the surface of endothelial cells.
- the invention therefore also relates to all molecules other than an anti-idiotype antibody capable of binding to the surface of endothelial cells using this receptor.
- the existence of such a receptor also makes it possible to use the HLA-Gls molecule, or these derivatives, to vectorize substances of interest at the level of angiogenic sites.
- the subject of the invention is therefore the use of the HLA-Gls molecule alone or in combination with a substance of interest for the preparation of a composition intended to inhibit angiogenesis or to detect angiogenic sites. As indicated previously, it may be the HLA-Gls molecule or a derivative thereof.
- the substance of interest can be a chemical or biological substance active like a drug.
- drugs that may be mentioned include: an anti-VEGF antibody, an anti-VEGF R2 antibody, an animal or plant toxin, a radioactive compound, a cytostatic or cytolytic drug, a suicide gene or an antisense.
- the substance of interest can also be a diagnostic substance such as a radioactive tracer or a contrast agent for magnetic resonance imaging making it possible to mark the angiogenic sites.
- the invention therefore relates to a compound consisting of the HLA-Gls molecule, or a derivative thereof, coupled to a substance of interest as defined above, said compound being capable of binding to endothelial cells by l through a receiver.
- These compounds of the invention therefore constitute a vectorisatid ⁇ system of substances of interest at the level of endothelial cells involved in angiogenic phenomena which are particularly useful for treating or detecting the pathologies indicated above.
- the coupling of the HLA-Gls molecule, or of a functional equivalent thereof, with the substance of interest can be carried out by any cleavable or non-cleavable bonding means in a biological medium such as: - coupling by covalent bond an oxidation reaction leading to the substitution of a hydrogen atom by an iodine atom on an indole nucleus
- These compounds of the invention can also be proteins or fusion genes.
- compositions according to the invention are particularly useful for the treatment of pathologies involving an excess of the proliferation of endothelial cells and requiring an inhibition of their growth.
- pathologies including treatment requires stimulation of angiogenesis, and endothelial proliferation, we can cicatrization, the maturation of the ovary yellow body, the reperfusion of ischemic areas during vascular thrombosis as in arteritis of the lower limbs and 1 ' myocardial infarction.
- compositions of the invention may be monoclonal or polyclonal, and prepared according to techniques well known to those skilled in the art by soluble molecules HLA class or a functional equivalent "thereof.
- the soluble HLA class 1 molecules used in the context of the invention can be prepared by conventional purification techniques as described in the experimental part below, but also by genetic engineering.
- the inventors produced the HLA-Gls molecule in a human eukaryotic system, in particular from human cells of trophoblastic origin (JAR line, Fournel et al., 1999).
- HLA-Gls Although the quantities of HLA-Gls produced by this system remain limited and that their production is carried out over a relatively long period, the advantages of this production system are as follows: (i), human ⁇ 2m is present in these cells and therefore associates in a natural non-covalent manner with the heavy chain HLA-Gls; (ii), the peptides which bind in the peptide pocket formed by the external domains l and ⁇ 2 of HLA-G are very probably identical to those present in vivo on the HLA-G1 molecules secreted by the cells of the extravillous cytotrophoblast during gestation and which must modulate the placental angiogenesis at the level of the decidua; (iii), the HLA-Gls molecule thus produced is naturally glycosylated when it would not be if a prokaryotic or insect cell system was used.
- Figure 2 shows the effect of the HLA-Gs incubation time on the phosphorylation of PLC- ⁇ .
- Figure 3 shows the effect of HLAGs on the phosphorylation of PI3K.
- FIG. 4 shows the effect of HLAGs on the phosphorylation of MAPK 42/44.
- FIG. 5 illustrates the inhibitory effects of the HLA-Gs molecule on the proliferation of endothelial cells in the presence of VEGF.
- FIG. 6 illustrates the modulation effects of ICAM-1 expression on HUVEC cells by flow cytometry.
- FIG 7 shows the results of tests for inhibition of the anti-chemotactic action of HLA GS or HLA B7 by the antibody W6 / 32.
- FIG. 8 shows the effects of HLA-Gs molecules on the tumor growth of PC3 cells transfected with HLA-Gs.
- FIG. 9 shows the results of the tests of inhibition of the action of HLA GS on the migration of endothelial cells by the antibody CL1-R2.
- HLA-Gls Purification of the soluble HLA-G1 molecule
- HLA-Gls The soluble HLA-G1 molecule (HLA-Gls) was purified in the INSERM U 395 laboratory from culture supernatants of HLA negative human eukaryotic cells, expressing ⁇ 2 -microglobulin ( ⁇ 2m), and transfected (stable transfections) with cDNA coding for the HLA-Gls heavy chain (containing intron 4): JAR cells of trophoblastic origin or .221 cells of lymphoid origin.
- the purification steps are as follows (Fournel et al., 1999):
- Elisa The presence of HLA-Gls molecules in the elution fractions is evaluated by Elisa (Fournel et al., 2000a).
- the positive Elisa fractions are pooled, concentrated and analyzed by SDS-PAGE or western blots using anti-HLA-G antibodies.
- HLA-Gs in the conditioned medium of clones of PC3 cells transfected with pcDNAl / HLA-Gs or pcDNAl is measured by the same ELISA technique.
- VEGF isoform of 165 amino acids
- the assay of VEGF in conditioned media of PC3 cells is carried out by test radio receiver according to the technique described above (Plou ⁇ t, 1990).
- the antibodies used are as follows:
- mouse anti-P-tyr antibody (Santa " Cruz Biotechnology PY20 cat sc-508) rabbit antibody anti PI3K (Upstate Biotechnology, p85 cat 06-195) - rabbit anti-p erk 1/2 antibody (New England
- the culture media and the sera come from the company Life Technologies TM .
- the endothelial cells of human umbilical cord veins (HUVEC) are cultured on plastic coated with gelatin diluted to 0.2% in PBS.
- the cells are cultured in endothelial medium SFM supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 20% of fetal calf serum and kept in an oven at 10% C02.
- SFM endothelial medium
- VEGF VEGF at 2 ng / ml added to the medium every two days.
- Fetal bovine aorta endothelial cells are cultured on plastic coated with gelatin diluted 0.2% in PBS.
- the cells are cultured in DMEM-glutamax medium supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 10% of newborn calf serum and kept in an oven at 10% C02.
- DMEM-glutamax medium supplemented with 50 units / ml of penicillin, 50 ⁇ g / ml of streptomycin, and 10% of newborn calf serum and kept in an oven at 10% C02.
- HUVECs are brought into contact with the modulators for 2 hours at 37 ° C. in a serum-free medium, then collected by scraping and washed twice with 0.2% PBS-BSA.
- HUVEC cells are grown in 12-well plates until subconfluence. The cell plates are placed on ice and washed three times with "binding buffer” (DMEM buffered to pH 7.38 with 20 mM HEPES, supplemented with 0.2% gelatin). They are then incubated for 3 hours at 4 ° C with a 1 ng / ml of VEGF or HLA-Gs iodized in a final volume of 500 ⁇ l. The non-specific binding of the iodinated ligand to the cells is determined in the presence of an excess of VEGF or
- HLA-Gs not iodized. After incubation, the cells are washed three times in "binding buffer” and then lysed with 500 ⁇ l of 0.5M NaOH. The radioactivity retained on the cells is measured using a gamma counter (Jonca, 1997). 7) Phosphorylations.
- HUVEC cells deprivated beforehand in serum are incubated with HLA-Gs in the presence or absence of VEGF during the period indicated in the figure) then washed twice with PBS supplemented with phosphatase inhibitors (ImM of sodium orthovanadate and sodium pyrophosphate) and protease inhibitors (2 ⁇ g / ml of each of the inhibitors aprotinin, leupeptin, pepstatin, benzamidine and 0.5M EDTA).
- the cell pellets are then lysed in RIPA buffer (0.1% SDS, 1% cacodylate, 1 mM EDTA, in 10 mM phosphate buffer, pH 7.4, supplemented with phosphatase and protease inhibitors).
- the lysates are clarified by centrifugation then the supernatant is preadsorbed on protein A sepharose beads (Pharmacia) for 1 hour at 4 ° C. After gentle centrifugation, the supernatant is incubated for two hours at 4 ° C with 2 ⁇ g / ml of each of the antibodies designated in the legend to the figure.
- the immune complexes are adsorbed on protein A sepharose beads, washed three times in lysis buffer and eluted in SDS-PAGE sample buffer. The eluted proteins are analyzed on an 8% gel (PLC gamma, PI3 kinase) or 10%
- MAP kinase of polyacrylamide.
- the gel is electrotransferred onto a nitrocellulose membrane.
- the membrane is then hybridized with the anti phosphotyrosine antibody PY 20 (Santa Cruz Biotechnology PY20 cat sc-508) diluted to l / 200 th then with a secondary anti mouse immunoglobulin antibody (1/10000) coupled with peroxidase (Amersham Life Science).
- PY 20 Anti phosphotyrosine antibody
- PY 20 Santa Cruz Biotechnology PY20 cat sc-508
- a secondary anti mouse immunoglobulin antibody (1/10000) coupled with peroxidase Amersham Life Science.
- the enzymatic activity is revealed by chemiluminescence with an ECL kit (Amersham Life Science) (Ortéga, 1997).
- FBAE or HUVEC cells are seeded at low density (1000 cells / cm2) in 12-well dishes, the surface of which has been previously coated with gelatin (Plou ⁇ t, 1989). Modulators are added after adhesion of the cells to the culture plastic, and after 2 days. Each condition is studied in three different wells. The cells are trypsinized and counted on the fifth day. The percentage of inhibition is calculated as the ratio (Ortéga, 1997):
- PC3 cells derived from prostatic adenocarcinoma were "" transfected with the vector " PCDNA containing the coding sequence of HLA-Gs (Fournel et al., 1999) or not (empty vector) by the use of lipofectamine previously described (Plou ⁇ t , 1997).
- the cells having integrated the geneticin resistance cDNA are selected with 600 mg / ml of G418 added to the culture medium.
- the cell clones were removed by aspiration using a micropipette under control.
- the cells from the sampled foci are then cultured and divided into several cultures, each of the cell clones is incubated with RPMI medium containing 5 mg / ml of insulin and transferrin for 48 h and the HLA-Gs and VEGF factors assayed.
- the screening of positive clones secreting HLA-Gs was carried out by Elisa and intracellular flow cytometry using a panel of specific anti-HLA-G antibodies (Fournel et al., 2000a).
- PC3 / Gsl6 are detached from the culture plastics using trypsin-EDTA, homogenized, centrifuged and suspended in culture medium supplemented with 10% fetal calf serum. 1 ml corresponding to 3.5 million cells, is injected under the skin of the right flank of each mouse. The viability of the suspension is checked beforehand by vital staining with trypan blue; the dye is excluded from more than 99% of the cells. 6 groups 7 female nude mice 6 weeks old were implanted.
- FIG. 1 illustrates the results of the study of factor binding to receptors by a competitive immunoassay, in which subconfluent HUVEC cells are incubated in binding buffer at 4 ° C. with 1 ng / ml of iodized factors as well as with non-iodized factors. After 4 hours, the cells are washed twice in PBS at 4 ° C. The cells are lysed in 0.2M NaOH and the bound radioactivity is measured in a gamma counter.
- Figure 2 shows the effect of the HLA-Gs incubation time on the phosphorylation of PLC- ⁇ .
- HUVEC are deprived in serum for 24 hours.
- the HLA-Gs is added for 10 or 60 minutes. 120 ng / ml of VEGF is then added or not for 15 minutes.
- the cells are washed twice at 4 ° C. with PBS in the presence of protease inhibitors and phosphatase inhibitors.
- the cells are collected by scraping in 5 ml of PBS and centrifuged at 400 g.
- the cell pellets are lysed in a RIPA buffer at 4 ° C., preadorbed on beads. protein A sepharose 1 hour then incubated two hours at 4 ° C with 2 ⁇ g / ml of anti-PLC- ⁇ antibody.
- the immune complexes are adsorbed on protein A sepharose beads and washed three times in lysis buffer.
- the supernatants are eluted, denatured and reduced in sample buffer containing 50 mM beta-mercaptoethanol and then analyzed on an 8% polyacrylamide gel.
- the gel is electrotransferred onto a nitrocellulose membrane which is then dehydrated and then hybridized with an anti-P-tyr monoclonal antibody and then revealed with a secondary antibody coupled to peroxidase.
- the enzymatic activity is revealed by chemiluminescence with an ECL kit
- Figure 3 shows the effect of HLAGs on the phosphorylation of PI3K.
- Six million HUVECs are deprived in serum for 24 hours.
- the HLA-Gs is added for 10 or 60 minutes.
- 120 ng / ml of VEGF is then added or not for 15 minutes.
- the cells are washed twice at 4 ° C. with PBS in the presence of protease inhibitors and phosphatase inhibitors.
- the cells are collected by scraping in 5 ml of PBS and centrifuged at 400 g.
- the cell pellets are lysed in RIPA buffer at 4 ° C. then denatured and reduced in sample buffer containing 50 mM beta-mercaptoethanol and analyzed on an 8% polyacrylamide gel.
- the gel is electrotransferred onto a nitrocellulose membrane which is then dehydrated and then hybridized with an anti PI3 K antibody and then revealed with a secondary anti rabbit IgG antibody coupled to peroxidase.
- the enzymatic activity is revealed by chemiluminescence with an ECL kit.
- the membrane is then dehybridized and re-hybridized with a polyclonal antibody directed against PI3 K.
- Figure 4 shows the effect of HLAGs on the phosphorylation of MAPK 42/44.
- One million HUVECs are deprived in serum for 24 hours.
- the HLA-Gs is added for 10 or 60 minutes.
- 120 ng / ml of VEGF is then added or not for 15 minutes.
- the cells are washed twice at 4 ° C. with PBS in the presence of protease inhibitors and phosphatase inhibitors.
- the cells are collected by scraping in 5 ml of PBS and centrifuged at 400 g.
- the cell pellets are lysed in RIPA buffer at 4 ° C. then denatured and reduced in sample buffer containing 50 mM beta-mercaptoethanol and analyzed on an 8% polyacrylamide gel.
- the gel is electrotransferred onto a nitrocellulose membrane which is then dehydrated and then hybridized with an anti p erkl / 2 antibody and then revealed with a secondary anti rabbit IgG antibody coupled to peroxidase.
- the enzymatic activity is revealed by chemiluminescence with an ECL kit.
- the membrane is then dehybridized and re-hybridized with a polyclonal antibody directed against erk 2.
- FIG. 5 illustrates the inhibitory effects of the HLA-Gs molecule on the proliferation of endothelial cells in the presence of VEGF.
- FBAE cells are seeded at low density (5000 cells / well) in 12-well dishes.
- Variable doses of HLA Gs or HLA B7 are added in the presence or absence of 2 ng / ml of VEGF after adhesion of the cells on the first and third day.
- the cells are trypsinized and counted. Each well is produced in triplicate. The percentage inhibition is calculated as described in the text.
- FIG. 6 illustrates the modulation effects of the expression of ICAM-1 on HUVEC cells by flow cytometry.
- HUVEC cells are placed in the presence of HLAG-s, HLAB-7 in the presence or absence of VEGF 2 hours at 37 ° C.
- 500,000 cells collected by scraping are incubated for 2 hours at 4 ° C with 2 ⁇ g / ml of anti-ICAM-1 antibody.
- an anti-mouse Fc antibody coupled to the 1/100 diluted FITC is then incubated for half an hour. Two washes in 0.2% PBS-BSA are again carried out, then the cells are analyzed by flow cytometry.
- Figure 7 illustrates the effects of the HLA-Gs molecule on the migration of endothelial cells. This experiment shows that the HLA-Gs and HLA B7s molecules have no effect on the spontaneous migration of cells. On the other hand, a molar excess of only 4 times is sufficient to almost completely inhibit the chemotactic activity of VEGF.
- Figure 7 further shows the results of inhibition assays of the action "anti-chemotactic HLA B7 or HLA GS by antibody W6 / 32.
- the FBAE cells are seeded at high density (40,000 cells / well) in 12-well dishes in the presence of 2 ng / ml of VEGF 2 days after confluence cell proliferation is stopped by transfer to a medium containing neither serum nor VEGF for 24 hours, an injury is then practiced in the monolayer by scraping using a foam tip, the cells rinsed thoroughly and then the modulators are added Each condition is studied in two different wells 24 hours after the cells are rinsed with DMEM medium, stained with May Grunwald-Giemsa and the migrated cells are counted in 8 fields by condition.
- FIG. 8 shows the effects of the HLAGs molecules on the tumor growth of the PC3 cells transfected with HLA-Gs.
- Six million PC3 cells transfected with the control vector (PC3-P3) or the vector containing the coding sequence of HLA Gs (clones PC3-Gsll, 15 and 16) are inoculated into 7 nude mice. After 20 days, the tumor volume is measured twice a week. The measurements are carried out by an operator ignoring the allocation of the mice in each of the 4 groups. On the 53rd day, the mice are sacrificed and the tumor is weighed. The doubling times were analyzed by the kinetics of the proliferation of each of the clones. The anti-HLAGs ELISA measurement was carried out with the antibody 87G.
- Figure 9 reports the identification of the endothelial receptor for soluble HLA G molecules.
- HLA-Gs and HLA B7s have no effect on spontaneous cell migration. On the other hand, a molar excess of only 4 times is sufficient to almost completely inhibit the chemotactic activity of VEGF.
- the antibody CL1-R2 directed against the BY55 receptor known to be a HLA-Gs receptor on NK lymphocyte cells, also inhibits the action of HLA-Gs on the migration of endothelial cells. Therefore BY55 is an endothelial receptor for HLA-Gs.
- Soluble HLA-G purification from eukaryotic transfected cells and detection by a specifies ELISA. Am J Reprod Immunol 42, 22-29.
- HLA-G human leukocyte antigen G
- HLA-G in human thymus subpopulation of medullary epithelial but not CD83 + dendritic cells expresses HLA-G as a membrane bound and soluble protein.
- HLA-Gl Mallet V., Prôll J., Solier C, Aguerre-Girr M., DeRossi M., Loke Y., Lieri F. and Le Bouteiller P.
- the full length HLA-Gl and no other alternative form of HLA-G is expressed at the cell surface of transfected cells.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Transplantation (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR01/10554 | 2001-08-07 | ||
FR0110554A FR2828403B1 (fr) | 2001-08-07 | 2001-08-07 | Utilisation de molecules solubles hla de classe 1 pour la preparation de compositions pharmaceutiques utiles pour inhiber l'angiogenese |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003018048A1 true WO2003018048A1 (fr) | 2003-03-06 |
Family
ID=8866352
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2002/002812 WO2003018048A1 (fr) | 2001-08-07 | 2002-08-06 | Utilisation de molecules solubles hla de classe 1 pour inhiber l'angiogenese |
Country Status (2)
Country | Link |
---|---|
FR (1) | FR2828403B1 (fr) |
WO (1) | WO2003018048A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1626059A1 (fr) * | 2004-08-09 | 2006-02-15 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Applications angiogéniques et immunologiques de composés spécifiques anti-CD160 pouvant être obtenus à partir de l'anticorps monoclonal CL1-R2 |
EP2210903A1 (fr) | 2009-01-21 | 2010-07-28 | Monoclonal Antibodies Therapeutics | Anticorps monoclonaux anti-CD 160 et leurs utilisations |
US8815235B2 (en) | 2004-06-02 | 2014-08-26 | Jan E. Schnitzer | Tissue-specific imaging and therapeutic agents targeting proteins expressed on lung endothelial cell surface |
JP5637981B2 (ja) * | 2009-03-03 | 2014-12-10 | 国立大学法人北海道大学 | 関節リウマチまたはその関連疾患の予防または治療剤 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2934498B1 (fr) * | 2008-08-01 | 2014-08-15 | Commissariat Energie Atomique | Utilisation d'une forme soluble d'hla-g dans le traitement des proliferations anormales des lymphocytes b. |
-
2001
- 2001-08-07 FR FR0110554A patent/FR2828403B1/fr not_active Expired - Fee Related
-
2002
- 2002-08-06 WO PCT/FR2002/002812 patent/WO2003018048A1/fr not_active Application Discontinuation
Non-Patent Citations (6)
Title |
---|
A. BLASCHITZ ET AL.: "Endothelial cells in chorionic fetal vessels of first trimester placenta express HLA-G.", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 27, no. 12, December 1997 (1997-12-01), pages 3380 - 3388, XP008010900 * |
FUJII T ET AL: "A SOLUBLE FORM OF THE HLA-G ANTIGEN IS ENCODED BY A MESSENGER RIBONUCLEIC ACID CONTAINING INTRON 4", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 153, no. 12, 15 December 1994 (1994-12-15), pages 5516 - 5524, XP002061945, ISSN: 0022-1767 * |
P. LE BOUTEILLER ET AL.: "Placental HLA-G protein expression in vivo: where and what for?", HUMAN REPRODUCTION UPDATE, vol. 5, no. 3, 1999, pages 223 - 233, XP001051115 * |
P. LE BOUTEILLER ET AL.: "The functionality of HLA-G is emerging.", IMMUNOLOGICAL REVIEWS, vol. 167, 1999, Copenhagen, DK, pages 233 - 244, XP008005346 * |
REBMANN V ET AL: "DETECTION OF SOLUBLE HLA-G MOLECULES IN PLASMA AND AMNIOTIC FLUID", TISSUE ANTIGENS, MUNKSGAARD, COPENHAGEN, DK, vol. 53, no. 1, January 1999 (1999-01-01), pages 14 - 22, XP000989657, ISSN: 0001-2815 * |
S. FOURNEL ET AL.: "Cutting edge: Soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8", THE JOURNAL OF IMMUNOLOGY, vol. 164, no. 12, 15 June 2000 (2000-06-15), BALTIMORE, US, pages 6100 - 6104, XP002204753 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8815235B2 (en) | 2004-06-02 | 2014-08-26 | Jan E. Schnitzer | Tissue-specific imaging and therapeutic agents targeting proteins expressed on lung endothelial cell surface |
EP1626059A1 (fr) * | 2004-08-09 | 2006-02-15 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Applications angiogéniques et immunologiques de composés spécifiques anti-CD160 pouvant être obtenus à partir de l'anticorps monoclonal CL1-R2 |
WO2006015886A1 (fr) * | 2004-08-09 | 2006-02-16 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Applications angiogénique et immunologique de composés d'anticorps-cd 160 spécifiques obtenus à partir de l’anticorps monoclonal cl1-r2 |
US8444978B2 (en) | 2004-08-09 | 2013-05-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Angiogenic and immunologic applications of anti-CD160 specific compounds obtainable from mAb CL1-R2 |
EP2210903A1 (fr) | 2009-01-21 | 2010-07-28 | Monoclonal Antibodies Therapeutics | Anticorps monoclonaux anti-CD 160 et leurs utilisations |
WO2010084158A1 (fr) | 2009-01-21 | 2010-07-29 | Monoclonal Antibodies Therapeutics | Anticorps monoclonaux anti-cd 160 et leurs utilisations |
JP5637981B2 (ja) * | 2009-03-03 | 2014-12-10 | 国立大学法人北海道大学 | 関節リウマチまたはその関連疾患の予防または治療剤 |
Also Published As
Publication number | Publication date |
---|---|
FR2828403B1 (fr) | 2003-10-17 |
FR2828403A1 (fr) | 2003-02-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ezekowitz et al. | Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells. | |
Waldmann | The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes | |
US7902151B2 (en) | Methods and compositions for modulating immunity | |
US5759536A (en) | Use of fas ligand to supress T-lymphocyte-mediated immune responses | |
CN1449445B (zh) | Eg-vegf核酸和多肽及使用方法 | |
US7553811B2 (en) | Methods and compositions for modulating immunity | |
Haspot et al. | Anti‐CD28 Antibody‐Induced Kidney Allograft Tolerance Related to Tryptophan Degradation and TCR− Class II− B7+ Regulatory Cells | |
US9018006B2 (en) | Stable Tregs and related materials and methods | |
JPH05509312A (ja) | 組織由来の腫瘍増殖インヒビター,その調製方法及び使用 | |
Hammer et al. | HLA class I expression on the materno‐fetal interface | |
US20120269806A1 (en) | Methods of inducing tolerance | |
US6749847B2 (en) | Hybrid cytokine of IL-7 and β-chain of hepatocyte growth factor | |
AU2001233502B2 (en) | Pancreatic islet cell growth factors | |
EP0868434B1 (fr) | Anticorps anti-idiotypiques du facteur de croissance endotheliale vasculaire et leur utilisation comme medicaments | |
JPH10512267A (ja) | 新規ペプチド | |
WO2003018048A1 (fr) | Utilisation de molecules solubles hla de classe 1 pour inhiber l'angiogenese | |
Blancho et al. | MOLECULAR AND CELLULAR EVENTS IMPLICATED IN LOCAL TOLERANCE TO KIDNEY ALLOGRAFTS IN MINIATURE SWINE1 | |
WO2000073427A2 (fr) | Peptides derives de l'acetylcholinesterase et leurs utilisations | |
US20070020703A1 (en) | Use of compositions comprising a soluble form of hla-g in the treatment of blood diseases | |
JP2000515001A (ja) | ダウンレギュレーション耐性c3コンバターゼ | |
FR2641468A1 (fr) | Anticorps chimeriques anti-recepteur de l'interleukine-2 humaine, leur preparation et compositions pharmaceutiques les contenant | |
Govallo | Immunology of pregnancy and cancer | |
Samlowski et al. | Nitric oxide exposure inhibits induction of lymphokine-activated killer cells by inducing precursor apoptosis | |
Longnecker et al. | Regulation of growth and differentiation in pancreatic cancer | |
Fusenig et al. | Cell culture in pharmaceutical research |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VN YU ZA ZM Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |