WO1994019021A1 - Conjugues proteiques, compositions les contenant et leurs applications en tant que medicament - Google Patents
Conjugues proteiques, compositions les contenant et leurs applications en tant que medicament Download PDFInfo
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- WO1994019021A1 WO1994019021A1 PCT/FR1994/000183 FR9400183W WO9419021A1 WO 1994019021 A1 WO1994019021 A1 WO 1994019021A1 FR 9400183 W FR9400183 W FR 9400183W WO 9419021 A1 WO9419021 A1 WO 9419021A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
Definitions
- PROTEIN CONJUGATES COMPOSITIONS CONTAINING THEM AND THEIR APPLICATIONS AS MEDICAMENTS.
- the present invention relates to protein conjugates with specific affinity towards appropriate target cells, and more particularly towards cancer cells, as well as to their applications as an anticancer drug.
- an amc is prepared so as to have specificity for an anti ⁇ gene present on the surface of the target cell, so that the amc will bind to the target cell, and when it is incorporated into said cell , it will transport its toxic conjugate inside the latter.
- the toxic conjugate is mostly ricin, or the A subunit of ricin.
- the B subunit binds to ubiquitous galactose residues on the cell surface, while the enzyme inactive A subunit enters the ribosomes. It has been estimated that a single castor molecule can inactivate all ribosomes of a cell. The invaded cell, unable to produce new proteins, dies.
- agents can be incorporated into immunotoxins; mention may be made, in particular of bacterial or plant toxins (or their active fragments), radioactive atoms, and agents used in anticancer chemotherapy.
- the immunotoxins thus formed direct the active substance (the toxin or the anticancer agent) towards the pathological domain and thus lead to the elimination of the target cells.
- the toxin / specific antibody conjugates form very active immunotoxins, but they also have non-specific toxicity which restricts their application in vivo.
- fragments of toxins devoid of sites capable of recognizing the targeted targets and conjugated to antibodies, are commonly used as immunotoxins.
- Such hybrid molecules such as for example those which contain a deglycosylated ricin-A chain, are thus made highly specific.
- their activity is lower than that of the immunotoxins comprising total ricin and this activity depends on the intensity with which the endocytosis takes place.
- the translocation of the immunotoxin in the target cell essentially depends on the surface anti ⁇ gene recognized by the antibody component of the immunotoxin. It is an important step in the action of immunotoxins which, in a large number of cases, does not lead to endocytosis; consequently, the intracellular translocation of the immunotoxin therefore does not occur. In such cases, treatment therefore results in failure. In addition, many anti-gene markers present on the surface of cells are secreted. also in the extracellular medium. The effectiveness of the immunotoxin is therefore very reduced because of the competitive effect between surface antigens and secreted antigens.
- cytotoxic agent is often problematic. Indeed, it must be relatively non-toxic once conjugated and become much more toxic after its transport to its site of action where intracellular enzymes release the toxic substance within tumor cells.
- the Applicant has therefore set itself the goal of providing a composition, for therapeutic purposes, capable of acting only at the level of specific targets (cancer cells) and capable of promoting endocytosis (stimulation of the transport of substances across cell membranes).
- the present invention relates to protein conjugates, of the type comprising a non-specific part and a part having a particular affinity for a specific type of target cells, characterized in that the part having a particular affinity for a specific type of target cells, (also called specific transport substance to the target cell) is ⁇ -fetoprotein and in that the non-specific part of said conjugate is chosen from the group which comprises the cytotoxic substances chosen from animal toxins, plant toxins, cytolytic enzymes and low molecular weight anti-cancer active principles selected from carboxyphosphoamide, amboclorin, doxorubicin, bleomycin, cisplatin, vinblastin, calichemycin and methotrexate or substances that modify the metabolism of cancer cells, such as nucleotide sequences d
- conjugates retain some of the properties of ⁇ -fetoprotein and are, in particular, easily incorporated into cells which carry the ⁇ -fetoprotein receptor on their surface, by an endocytosis mechanism which stimulates penetration of the non-specific part (cytotoxic substance, nucleotide sequence) and are only active vis-à-vis cancer cell lines such as ly phomas, hepatomas, neuroblas omes, melanomas, astrocytomas, teratoblastomas, embryonic carcinomas, ovaries , testicular or presacral.
- endocytosis mechanism which stimulates penetration of the non-specific part (cytotoxic substance, nucleotide sequence) and are only active vis-à-vis cancer cell lines such as ly phomas, hepatomas, neuroblas omes, melanomas, astrocytomas, teratoblastomas, embryonic carcinomas, ovaries , testicular or presacral.
- the conjugate has, with respect to the target cells, an activity significantly higher than that obtained with the non-specific non-conjugated part.
- the present invention also relates to pharmaceutical compositions, characterized in that they comprise, as active principle, at least one protein conjugate according to the invention and at least one pharmaceutically acceptable vehicle.
- ⁇ -fetoprotein-cytotoxic protein conjugates chosen from the anti-cancer active principles are particularly suitable for being used for the preparation of a medicament intended for use in the treatment of cancers and in particular in the treatment of leukemias , lymphomas, hepatomas, neuroblastomas, melanomas and astrocytes.
- the conjugates ⁇ -fetoprotein-vinblastine, ⁇ -fetoprotein-doxorubicin and ⁇ -fetoprotein-calichemicin are particularly suitable for being used for the preparation of a medicament intended for use in chronic myeloid leukemia.
- the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.
- Amounts of ⁇ -fetoprotein, of the order of a milligram can be prepared by immunoaffinity techniques.
- the first step therefore consists in preparing this fairly pure protein and in sufficient quantity (approximately 100 ⁇ g) to immunize mice and to prepare monoclonal antibodies which will then be used to purify the ⁇ -fetoprotein in significant quantities. one step.
- the ⁇ -fetoprotein can be prepared from blood from the human fetal cord or from cancerous cells in culture (hepatomas, teratocarcinoma).
- the production method consists, in fact, of separating this protein from the other proteins which accompany it, according to conventional methods known per se, such as ion exchange, affinity, exclusion chromatography on gel or hydrophobic interaction, preparative electrophoresis, dialysis and ultrafiltration.
- the raw material whole blood or cell suspension
- the total proteins are then purified by chromatography and then by preparative electrophoresis, so as to avoid any contamination by foreign proteins and in particular by albumin.
- the eluted proteins are collected and then chromatography is carried out on a Mono Q column.
- PHARMACIA FPLC SYSTEM Elution is carried out by a sodium chloride gradient (0-0.5 M) in the same buffer as above. The ⁇ -fetoprotein is eluted with 0.35 M of sodium chloride. After concentration by ul ⁇ trafiltration on a PM-10 membrane with a cutoff threshold of 10,000 daltons (AMICON), the proteins are chromatographed again by filtration on crosslinked agarose gel (Superose® 12, PHARMACIA) , in a column 25 mm in diameter and 80 cm long in a pH 7.0 phosphate buffer. The column having been previously calibrated, the peaks corresponding to a molecular weight of 70 Da and 140 kDa are collected.
- the electrophoresis is carried out without SDS, from a concentration gel zone (4% polyacrylamide in the concentration gel and 7.5% in the separation gel).
- a Tris-HCl buffer pH 6.7 is used in the concentration gel and a Tris-glycine buffer pH 8.3 in the separation gel.
- the sample is diluted to half with Tris-HCl buffer pH 6.7.
- Tris-HCl buffer pH 6.7 One part of glycerol is added for one part of sample diluted in the buffer, and one-tenth of the volume to be deposited of bromophenol blue solution is added in Tris-HCl pH 6.7 buffer, to visualize the front of migration.
- Electrophoresis is carried out at 15 "C at 30 mA for about 4 hours.
- Coomassie and the strip in its uncoloured part correspond- laying on the ⁇ -fetoprotein, is carefully cut to avoid contamination with the albumin still present at this stage of the purification.
- the gel containing the ⁇ -foeto ⁇ protein is homogenized in pH 7.0 phosphate buffer and stirred in a tube for 1 hour.
- the supernatant is collected and chromatographed on Cibacron Blue 2-Sepharose® as before, but in phosphate buffer pH 7.0. Proteins not retained by the gel are collected and then concentrated by ultrafiltration, as above. These proteins are then used to immunize mice in order to obtain antibodies which will make it possible to prepare significant amounts of ⁇ -fetoprotein by immunoaffinity.
- the ⁇ -fetoprotein is used to produce antibodies of low affinity anti- ⁇ -fetoprotein. This production occurs by injecting ⁇ -fetoprotein into mice in the presence of Freund's adjuvant.
- the popliteal lymph nodes are then removed from these mice and the cells of these lymph nodes are fused with myeloma cells to form hybridomas.
- the immunization period is voluntarily chosen to be very short (one week) contrary to the usual methods which extend over several months.
- mice This method makes it possible to produce clones of anti- ⁇ -fetoprotein antibodies of low affinity. More specifically, two BALB / c mice, about 4 months old, are immunized by injection of 20 ⁇ g of proteins associated with Freund's adjuvant, in a hind paw, twice to two days before , then a third time with 50 ⁇ g of protein without adjuvant, two days later. Three days after the last injection, or nine days in all, the mice are sacrificed and the popliteal lymph nodes are removed. They are homogenized and washed with DMEM medium without fetal calf serum and the homogenate is used to fuse with Sp2 / o myeloma cells. These mouse myeloma cells are conventionally used to produce hybridomas, they have the property of not developing in the HAT medium. Sp2 / o cells are cultured on DMEM- medium
- Hybrimax with 10% fetal calf serum The fusion is carried out in the presence of PEG 3,000, at a concentration of 50% in DMEM medium from Fl thymocytes
- Hybridomas are grown in HAT medium. The clones appear in 10-15 days in 75% of the cultures and correspond to Sp2 / o supplemented by immunocompetent cells.
- the monoclonal antibodies of the IGg type are then purified by chromatography on Protein-A- Sepharose® (PHARMACIA). 5 ml of ascites liquid are centrifuged at 3000 rpm for 20 minutes, half diluted with Tris-HCl, pH 8.1, and placed on a column of Protein-A-Sepharose®. The column is washed with 100 ml of Tris-HCl buffer, pH 8, 1, and the immunoglobulins are eluted with an acetate buffer pH 6.0, then pH 4.5 and finally pH 3.5.
- the low affinity anti- ⁇ -foeto ⁇ protein antibodies of one of the clones (10C3) are eluted at pH 4.5 and another (7H8) at pH 3.5.
- the immunoglobulins are then concentrated by precipitation with ammonium sulfate at 50%, at 4 ° C for 18 hours and centrifuged for 30 minutes at 4000 rpm. The centrifugation pellet is redissolved in a minimum volume of PBS buffer and dialyzed against the same buffer, for 24 hours. 3 to 5 mg of antibody are thus obtained for 1 ml of ascites fluid taken from the mice.
- the immunoglobulins thus purified are then immobilized on a Sepharose® gel (PHARMACIA) activated by cyanogen bromide according to the method pre ⁇ approved by the supplier of the gel.
- PHARMACIA Sepharose® gel
- a column 25 mm in diameter and 50 cm long is prepared with Sepharose gel to which the monoclonal antibody is fixed as described above.
- the column is loaded with 50 ml of human fetal serum and the ⁇ -fetoprotein is eluted with a phosphate buffer in the form of a pH gradient ranging from 3.5 to 6.0.
- the ⁇ -fetoprotein is eluted at pH 6.0, in a proportion of approximately 90%.
- the ⁇ -fetoprotein is a glycoprotein.
- the two domains of the molecule, protein and osidic can be used as binding sites for different substances.
- Any substance having a chemically active site can, a priori, be conjugated to the ⁇ -fetoprotein.
- the molecules containing thiol residues can be linked to the ⁇ -fetoprotein by a reaction between amino groups of the protein and activated carboxylic esters or with imidates.
- the fixing of molecules containing carboxylic residues is effected in a particularly effective manner by derivatives of carbodiimides. In general, the fixation on osidic residues is effected by modification with sodium metaeriodate, in particular when it is a question of fixing molecules containing free amino residues.
- Targets for the conjugated ⁇ -fetoprotein Any cell having on its surface the receptor for the ⁇ -fetoprotein is a possible target for the conjugated ⁇ -fetoprotein. No normal cells or non-fetal has this receptor. Only fetal cells and cancer cells are provided with a proportion which exceeds 90% and this in more than 70 different cases of cancer (R. MORO et al., Tumor Biol., 8, 293, 1987).
- EXAMPLE 2 Preparation of conjugates in accordance with the invention a) ⁇ -fetoprotein-toxin - ⁇ -fetoprotein (AFP) and diphtheria toxin (DT) conjugates.
- AFP ⁇ -fetoprotein-toxin - ⁇ -fetoprotein
- DT diphtheria toxin
- diphtheria toxin 10 mg are placed in an aqueous solution containing 50 mM of dithiothreitol and incubated for 1 hour at room temperature.
- the protein thus reduced is then purified by chromatography on a column of Sephadex® G-25 equilibrated in PBS buffer, or dialysis against the same buffer.
- the reduced toxin is brought into contact with the modified ⁇ -fetoprotein and the solution of the two proteins is concentrated by ultrafiltration on a cellulose membrane having a cutoff threshold of 10,000 daltons. It is then left to incubate for 18 hours at room temperature.
- the product obtained is then purified by chromatography on a column of Sephacryl® S-3.0 (PHARMACIA).
- the final yield of the conjugation is 34%.
- AFP ⁇ -fetoprotein
- R ricin A
- ⁇ -fetoprotein 1 mg is dissolved in 1 ml of PBS buffer and mixed with 6 mg of SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate) in ethanolic solution at a rate of 1 mg / ml. The mixture is incubated for 30 minutes, at room temperature, with constant agitation. The ⁇ -fetoprotein thus modified is dia ⁇ lysee against a 0.05 M borate buffer, pH 8.5.
- SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
- ricin A is treated under the same conditions and in the same quantities as 1 ⁇ -fetoprotein.
- the two modified proteins are then brought into contact, concentrated by ultrafiltration on a cellulose membrane with a cutoff threshold of 10,000 daltons up to a final volume of 5 ml, and incubated for 18 hours at room temperature.
- the result of the conjugation thus produced is then purified by filtration chromatography on Sephacryl® S-300 gel.
- the conjugate is recovered in its buffer pH 8.5, at the molecular weight corresponding to the sum of the molecular weights of the two proteins, ie approximately 102,000 daltons.
- AFP - ⁇ -fetoprotein
- Asp asparaginase
- CMC l-cyclohexyl-3 (2-morpholinoethyl) carbodiimide
- Human AFP (Asp: AFP molar ratio of 1: 1) is added to a solution of asparaginase, activated by an excess, by a factor of the order of 2000, of CMC, at pH 5.4. The mixture obtained is incubated for 16 hours at room temperature and dialyzed against PBS.
- Carboxyphosphoamide is the metabolite of cyclophosphoamide, a widely used anticancer molecule. Its metabolite, carboxyphosphoamide, cannot penetrate cells because of its high negative charge, and thus manifests low toxicity. If, by means of conjugation, carboxyphosphoamide enters the cells, it will be split into acrolein and phosphoramide by phosphoamidases which are particularly active in cells in the process of proliferation. The phosphoramide thus obtained is highly cytotoxic.
- the conjugation between the ⁇ -fetoprotein and the carboxyphosphoamide is carried out at + 4 "C in pyridine containing 0.01 M HCl and EDC (1-ethyl-3 (3-dimethyl-aminopropyl ) carbodiimide) in an EDC: AFP proportion of 2500: 1.
- EDC Hexamethylenediamine
- HMDA Hexamethylenediamine
- the final ⁇ -fetoprotein-hexamethylene-diamine-CFA proportions are 1: 10: 120
- the resulting conjugate is dialyzed against PBS and then stored at -70 ° C.
- N (CH2-CH2 ⁇ C1) 2 can be conjugated to the ⁇ -fetoprotein in the same way as carboxyphosphoamide, with a yield of approximately 65%.
- Methotrexate (MT) is dissolved in a pyridine-HCl buffer, pH 5.0 and the ⁇ -fetoprotein (AFP) solution is prepared in the same buffer. EDC is added and the whole is agitated.
- MT AFP: EDC is 10: 1: 2500. After shaking for 3 hours, the preparation is dialyzed against a PBS buffer and diluted in PBS until a concentration of AFP of 20 mg / ml is obtained.
- DR doxorubicin
- HC1, pH 5.0 and the ⁇ -fetoprotein (AFP) solution is prepared in the same buffer.
- EDC and adipic acid are added.
- Adipic acid plays the role of intercalator between AFP and DR; the DR: adipic acid: AFP: EDC ratio is 10: 50: 1: 2500.
- Daunomycin has a free amino radical which allows direct conjugation with ⁇ -fetoprotein. Daunomycin is dissolved in pyridine at a concentration of 10%. A solution of ⁇ -fetoprotein in a hydrochloric buffer pH 5.0 is added, then adipic acid is added as an intercalator so that the proportions between the different products are variable but so that the ratio between ⁇ -fetoprotein and EDC is always from 1 to 2500.
- the yield of the conjugation strongly depends on the different proportions between the products and ranges from 22% to 57%.
- AFP ⁇ -fetoprotein
- RM rubomycin
- AFP-RM conjugates are prepared according to the method used for doxorubicin.
- AFP-BM conjugates are prepared using a method similar to that used for doxorubicin
- AFP - ⁇ -fetoprotein
- CP cisplatin
- AFP is added to a solution of cisplatin and EDC in 0.1 M pyridine buffer - HCl, pH
- the reaction mixture is stirred overnight at 4 ° C or 2 hours at 20 ° C.
- the conjugate obtained is dialyzed 3 times against a PBS buffer at 4 ° C, for 24 hours. Conjugate samples can be stored in the freezer.
- the EDC: AFP molar ratio must not be less than 2000: 1.
- AFP - ⁇ -fetoprotein
- VB vinblastine
- Vinblastine does not have a free amino group; consequently, it is necessary to esterify it before carrying out a conjugation with AFP.
- the esterification is carried out, at 37 ° C., by dropwise addition of 0.1 M KOH to a solution of vinblastine, until a pH of 9.5 is obtained.
- the reaction mixture is neutralized with a 0.1 M pyridine-HCl solution at pH 5.0.
- the vinblastine thus modified is conjugated to AFP, according to a method similar to that described above for cisplatin; however, for the application of this method to vinblastine, EDC is introduced last, since vinblastine interacts with the amino groups of AFP.
- AFP - ⁇ -fetoprotein
- calichrobcine is first cleaved, by addition of dithiothreitol (up to 1 M) and AFP is modified by SPDP in PBS, pH 7.2, at 4 ° C , for 12 hours.
- the solutions of calichrobcine, EDC and modified AFP are mixed with stirring overnight.
- the conjugate obtained is dialyzed against a PBS buffer, for 24 hours.
- the SPDP: AFP molar ratio depends on the calichemycin: AFP ratio sought, and must be greater by a factor of 8 than the latter.
- EXAMPLE 3 Effects of the various conjugates in accordance with the invention on cultured human cancer cell lines.
- QOS QOS T lymphoma
- CEM CEM T lymphoma
- Raji B lymphoma Raji B lymphoma
- Namalva B lymphoma Nam
- Hep HepG2a hepatoma
- Amastrocytoma Ast
- Melanoma Bro Bro
- IMR neuroblastoma IMR - 32
- the aforementioned culture medium can also be adapted to each specific culture.
- Tables I to XI below which provide the percentage of surviving cells after an incubation of 72 hours with the cytotoxic substance alone (control) or with a conjugate in accordance with the invention (test).
- AFP diphtheria toxin
- AFP - ⁇ -fetoprotein
- R ricin A
- CFA cytotoxicity of the ⁇ -fetoprotein coupled to carboxyphosphoamide is determined with variable proportions of HMDA- ⁇ -fetoprotein of between 0 and 120. This determination is carried out on QOS cells, belonging to a lymphoblastoid line, by measuring the quantity of tritiated thymidine incorporated relative to the controls ( ⁇ -fetoprotein (AFP) alone, carboxy- phosphoamide (CPA) and HMDA alone or as a mixture in the same proportions as in the conjugate).
- AFP ⁇ -fetoprotein
- CPA carboxy- phosphoamide
- HMDA % of incorporated tritiated thymidine
- AFP - ⁇ -fetoprotein
- AC amboclorin
- AFP - ⁇ -fetoprotein
- DN daunomycin
- test a is carried out on the same cells and under the same conditions as for the ⁇ -fetoprotein conjugated to the carboxypnosphoamide (test a)). 1. Without interlayer
- Concentration Molar proportion Effect Effect in DN in DN AFP DN-AFP DN the medium
- Concentration Molar proportion Effect Effect in DN in DN AFP DN-AFP DN the medium
- AFP - ⁇ -fetoprotein
- RM rubomycin
- AFP - ⁇ -fetoprotein
- CP cisplatin
- AFP - ⁇ -fetoprotein
- VB vinblastine
- AFP ⁇ -fetoprotein
- CCh calichrobcine
- Type CCh AFP CCh AFP: CCh AF ?: CCh AFP: CCh AFP: from (24 CCh (48 CCh (60 CCh (240 CCh (24 CCh cel. PM) Z. - pM): 4 pM): 1 pM) ) 1: 2 nM) 1: 2
- the samples obtained are incubated at 37 ° C for 30-45 min, to allow the sedimentation of erythrocytes.
- the plasma is removed and the quantity of lymphocytes is estimated.
- the plasma is transferred to an RPMI medium, in the presence of various antibiotics (penicillin, gentamycin).
- the quantity of lymphocytes should not exceed 2.10 ⁇ / ml. If there are large amounts of leukemia cells in the sample, fetal bovine serum is added.
- Plasma Lymphocytes Plasma Lymphocytes total (24 h) total (48 h) (24 h) (48 h)
- the inhibitory effects are respectively a factor of about 45 and 16.
- the inhibitory effect of conjugates is less pronounced than for patient 1; this is due to the type of leukemia, the age and the previous treatments of the patient.
- the inhibitory effect of the conjugates is significantly more pronounced than that obtained with DR or CCh, alone.
- P388 or L1210 leukemia is transmitted by injection of 10 7 suitable cells to male DBA mice weighing approximately 18 g.
- the second day after the injection when the tumor has a quail of approximately 0.01 cm ⁇ - 1 , the treatment is started and includes the SC injection of an AFP-CCh conjugate (AFP: CC ratio. 1: 4), in accordance with the invention or calico-chemine alone (CCh at a dose of 0.6 ⁇ g / kg).
- AFP-CCh conjugate AFP: CC ratio. 1: 4
- calico-chemine alone CCh at a dose of 0.6 ⁇ g / kg.
- Anti-cancer preparations are introduced in SC, in the region of the tumor or in IV, (once / day for 7 days).
- a first group of animals used for control did not receive any preparation; a second group of animals also serving as controls received 0.2 ml of a 0.9% NaCl solution, in SC.
- Each group includes 6 animals.
- Monitoring of the size of the tumor is also carried out.
- Tables XV, XVI and XVII below:
- Agent Type Admin route Tumor volume
- Table XV shows that both in P388 leukemia and in L1210 leukemia, a significant decrease in tumor volume is observed in the treated animals. These results confirm the interest of the conjugates according to the invention.
- Table XVI shows the advantage of administering a conjugate according to the invention in SC.
- P388 or L1210 leukemia is transmitted by injection of 10 'suitable cells to male DBA mice weighing approximately 18 g.
- the treatment is started and includes SC injection of VB or AFP-VB conjugate (VB at a dose of 200 ⁇ g / kg, the AFP: VB ratio being 1:10) in the region of the tumor (1 fcis / day for 7 days) .
- a first group of animals used for control did not receive any preparation; a second group of animals also serving as control received 0.2 ml of 0.9% NaCl solution, in SC.
- Each group includes 6 animals.
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Abstract
Description
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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JP6518710A JPH08509698A (ja) | 1993-02-18 | 1994-02-18 | 蛋白質複合体、それを含む組成物及びその医薬としての用途 |
EP94907594A EP0684841A1 (fr) | 1993-02-18 | 1994-02-18 | Conjugues proteiques, compositions les contenant et leurs applications en tant que medicament |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR9301833A FR2701710B1 (fr) | 1993-02-18 | 1993-02-18 | Conjugués protéiques, compositions les contenant et leurs applications en tant que médicament et réactif de diagnostic. |
FR93/01833 | 1993-02-18 |
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WO1994019021A1 true WO1994019021A1 (fr) | 1994-09-01 |
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PCT/FR1994/000183 WO1994019021A1 (fr) | 1993-02-18 | 1994-02-18 | Conjugues proteiques, compositions les contenant et leurs applications en tant que medicament |
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EP (1) | EP0684841A1 (fr) |
JP (1) | JPH08509698A (fr) |
CA (1) | CA2155953A1 (fr) |
FR (1) | FR2701710B1 (fr) |
WO (1) | WO1994019021A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1996009551A1 (fr) * | 1994-09-19 | 1996-03-28 | Moro Ricardo J | Detection et traitement du cancer |
EP0712313A1 (fr) * | 1993-07-09 | 1996-05-22 | Alexei Kondratyev | Conjugues de medicaments pro-cytotoxiques destines a la therapie contre le cancer |
US5926113A (en) * | 1995-05-05 | 1999-07-20 | L & H Company, Inc. | Automatic determination of traffic signal preemption using differential GPS |
EP3659307A4 (fr) * | 2017-07-28 | 2021-09-22 | Yale University | Médicaments anticancéreux et leurs procédés de fabrication et d'utilisation |
Families Citing this family (2)
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US6635740B1 (en) | 1997-03-27 | 2003-10-21 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Ligand/lytic peptide compositions and methods of use |
US6680058B1 (en) | 1997-09-03 | 2004-01-20 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Compositions and methods for contraception in or sterilization of mammals |
Citations (1)
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WO1993005774A1 (fr) * | 1991-09-25 | 1993-04-01 | Wisconsin Alumni Research Foundation | Complexes d'antibiotiques d'anthracycline et d'acides gras polyinsatures dans des emulsions de lipides |
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US4195073A (en) * | 1977-10-27 | 1980-03-25 | Hoffmann-La Roche Inc. | Radioimmunoassay of alpha 1 fetoprotein |
US4894348A (en) * | 1987-07-01 | 1990-01-16 | Ronald Robert C | Fluorescein-conjugated proteins with enhanced fluorescence |
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1993
- 1993-02-18 FR FR9301833A patent/FR2701710B1/fr not_active Expired - Fee Related
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1994
- 1994-02-18 EP EP94907594A patent/EP0684841A1/fr not_active Withdrawn
- 1994-02-18 CA CA002155953A patent/CA2155953A1/fr not_active Abandoned
- 1994-02-18 WO PCT/FR1994/000183 patent/WO1994019021A1/fr not_active Application Discontinuation
- 1994-02-18 JP JP6518710A patent/JPH08509698A/ja active Pending
Patent Citations (1)
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WO1993005774A1 (fr) * | 1991-09-25 | 1993-04-01 | Wisconsin Alumni Research Foundation | Complexes d'antibiotiques d'anthracycline et d'acides gras polyinsatures dans des emulsions de lipides |
Non-Patent Citations (5)
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0712313A1 (fr) * | 1993-07-09 | 1996-05-22 | Alexei Kondratyev | Conjugues de medicaments pro-cytotoxiques destines a la therapie contre le cancer |
EP0712313A4 (fr) * | 1993-07-09 | 1998-10-07 | Alexei Kondratyev | Conjugues de medicaments pro-cytotoxiques destines a la therapie contre le cancer |
WO1996009551A1 (fr) * | 1994-09-19 | 1996-03-28 | Moro Ricardo J | Detection et traitement du cancer |
AU714966B2 (en) * | 1994-09-19 | 2000-01-13 | Ricardo J Moro | Detection and treatment of cancer |
EP1955714A1 (fr) * | 1994-09-19 | 2008-08-13 | Ricardo J. Moro | Détection et traitement du cancer |
EP1956374A1 (fr) * | 1994-09-19 | 2008-08-13 | Ricardo J. Moro | Détection et traitement du cancer |
US5926113A (en) * | 1995-05-05 | 1999-07-20 | L & H Company, Inc. | Automatic determination of traffic signal preemption using differential GPS |
EP3659307A4 (fr) * | 2017-07-28 | 2021-09-22 | Yale University | Médicaments anticancéreux et leurs procédés de fabrication et d'utilisation |
Also Published As
Publication number | Publication date |
---|---|
FR2701710A1 (fr) | 1994-08-26 |
CA2155953A1 (fr) | 1994-09-01 |
FR2701710B1 (fr) | 1995-04-21 |
EP0684841A1 (fr) | 1995-12-06 |
JPH08509698A (ja) | 1996-10-15 |
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