Classifications

• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/303—Liver or Pancreas
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/81—Packaged device or kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/804—Radioisotope, e.g. radioimmunoassay
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/806—Electrical property or magnetic property
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
  • Y10S436/808—Automated or kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
  • Y10S436/813—Cancer

Definitions

• ACINOFOETAL DIFFERENTIATION PROTEINS ASSOCIATED WITH PANCREAS CANCER, ANTISERUM AND ONOCLONAL ANTIBODIES AGAINST THESE PROTEINS, METHODS OF PREPARATION.
• the present invention relates to acino-fetal differentiation proteins associated with pancreatic cancer, their purification, a specific antiserum and its preparation, monoclonal antibodies specific for these proteins and their preparation process as well as the compositions for diagnostic use and for therapeutic use containing them.
• Panereatic oncofetal antigen (P0A-1) (banwo O., Versey J. and Hobbs JR, New ncofetal antigen for human pancreas. Lancet, 1: 643-645, 1974; Hobbs JR, Knapp, ML and Branfoot AG Panereatic oncofetal antigen (POA): its frequency and localization in humans. Oncod. Biol. And Medicine, 1: 37-48, 1980), 800 and 900 KD (POA-2) (Gelder FB , Resse CJ, Moossa AR, Hall T. and Hunter R. Purification partial characterization and clinical evaluation of a pancreatic oncofétal antigen.
• the present invention relates to proteins of differention-fetus acion associated with pancreatic cancer, characterized in that they are manosid glycoproteins, of apparent molecular ⁇ pyenne mass chosen from: 120 KD, 94 KD,
• the present invention also relates to a method
• the process for preparing these antigens includes
• a homogenate of soluble human fetal pancreas is prepared, b) the homogenate is treated by chromatography on an affinity column on concanavalin A coupled to a gel
• the proteins in question are eluted, and d) optionally, the proteins obtained are purified.
• the preparation of the homogenate is described in the example
• Sepharose gel 4B was used coupled with concanavalin A, marketed by Pharmacia. The unbound fraction is eliminated, the fixed fraction is eluted, using a specific ligand, ar
• the present invention also relates to an antiserum against the antigens according to the invention.
• this antiserum recognizes, in fetal pancreas extracts, 4 constituents of average apparent molecular mass 120, 94, 75 and 58 KD (to within 10%).
• the present invention relates to spreading a process for the preparation of this antiserum comprising the following steps: a) an animal is injected with an extract of human fetal pancreas less than 6 months old, b) the animal is bled and an antiserum is recovered , c) this antiserum is decomplemented, d) the decomplementary antiserum is absorbed, e) the absorbed decomplementary antiserum is centrifuged, and f) the specific antiserum is recovered.
• the animal is injected with an extract of the fetal pancreas less than 5 months old.
• This antiserum is prepared according to the protocol described in Example 2 against crude pancreatic extracts prepared as described in Example 1. In the particular embodiment described in Example 1, only the grooves No. 4 and 5 are positive in an antibody test. In one embodiment of the invention, the extract was injected into a rabbit. Mice and hens, for example, can also produce antiserum against these extracts.
• the antiserum To decomplement the antiserum, it is heated in a water bath at about 56 ° C for 1/2 hour, according to a conventional technique. The antiserum is then made specific to the fetal pancreas by absorption of various extracts, for example extracts from normal adult pancreas, normal serum, red blood cells A, O, B, and possibly other organs.
• pancreas comes from donor young people who died accidentally.
• the decomplemented antiserum absorbed is purified by a conventional centrifugation technique.
• the purified absorbed antiserum was tested for its ability to detect antigens by two techniques:
• NEF technique This technique is described in 1 * example 3. It makes it possible to detect very small quantities of one or more antigens in complex mixtures, for example extracts gross organs, pathological seru s. This technique can detect approximately up to 0.1 ⁇ g of differentiation proteins per ml in crude organ extracts or body fluids.
• the sensitivity varies with the antigen-antibody system and can go up to 0.01 ⁇ g of antigens per ml, or 0.0005% when, as for the tests collected in table 1, the extracts are analyzed from 20 mg / ml.
• the antiserum was positive for fetal pancreatic extracts, pancreatic tumors and liver metastases from primary pancreatic cancer.
• other extracts from adult organs were negative. or fetuses detailed in Table I.
• the extracts are "pools" of organs used in decreasing concentration from 20 mg / ml.
• the sera are used at lg2 dilutions from the normal concentration.
• Chronic panereatitis 11 Chronic pancreatitis is a special case because they are generally negative, but in some cases we observe a marking similar to that found in the p ritumoral areas.
• the antiserum according to the invention therefore makes it possible to detect serum and tissue levels which are too low to be assayed by precipitation, the sensitivity threshold of which is around 10 ⁇ g / ml.
• the Applicant has demonstrated that the proteins according to the invention appear not only in pancreatic tumors, but also in pretumoral lesions. These differentiation proteins therefore also have the value of pretumoral markers.
• the Applicant has carried out the preparation of monoclonal antibodies specific for the differentiation proteins according to the invention.
• the present invention relates to monoclonal antibodies specific for proteins of apparent molecular mass differentiation 120 KD, 94 KD, 75 KD and 58 KD.
• the present invention also relates to a process for the preparation of these monoclonal antibodies, characterized in that it comprises the following steps: a) a mouse is immunized with at least one of the differentiation proteins, b) a cell fusion of cells is carried out spleen of these mice, with an SP ⁇ myeloma, c) the hybridomas obtained are cultured in an appropriate medium, d) the cultures are screened, e) the hybridomas positive for screening are cloned and cloned specifically with the fetal pancreas, and, f) isolating and optionally purifying the monoclonal antibody.
• the conventional hybridization technique is therefore used, as developed by Kohler and Milstein in 1975, by immunizing mice, for example BALB / C mice with the fraction of a homogenate of fetal pancreas. under 6 months purified on Concanavalin A previously prepared.
• differentiation proteins prepared according to the invention were used to immunize the mice from fetal pancreas 3 to 4 months old.
• the NIF technique is used for example. The cultures are tested with various extracts, for example extracts from normal adult pancreas, normal adult and fetal serum, and an extract from fetal pancreas, in order to keep only the cultures which react specifically with the fetal pancreas. .
• the monoclonal antibodies are then selected by conventional techniques. It is thus possible to use the "Immuno Blot” technique using SDS-PAGE (example 5), a technique also known by the name of "Western Blot” and described by Towbin et al. (Electrophoretic Transfer of proteins from polyacrylamides gels to nitrocellular sheets procedure and some application, Prot. Nat. Acad. Sci. USA 7_, 1979 page 350-354).
• the monoclonal antibodies are then isolated in the supernatant of the cultures of selected hybridomas, by a usual technique.
• Monoclonal antibodies can also be obtained from ascites by injecting the producing cell, monoclonal antibody, into mice.
• the Applicant has thus isolated two monoclonal antibodies specific for the differentiation protein according to the invention with an average molecular mass of 120 KD.
• the antibodies can then be isolated by affinity chromatography on protein A fixed on sepharose, for example, eluting at a pH between 3 and 6.
• the present invention therefore also relates to compositions for diagnostic use containing, as active element, at least one differentiation protein or a monoclonal antibody according to the invention and an acceptable substrate, for example in the form of immunodiagnostic kits. according to a mechanism based on antibody-antigen reactions.
• the differentiation proteins and monoclonal antibodies according to the invention are also useful as specific tumor and pretumoral markers which can be used for imaging.
• compositions for diagnostic use comprising the differentiation proteins or the monoclonal antibodies according to the labeled invention.
• the labeling can be carried out either by any marker, in particular fluorescent, radioactive or paramagnetic markers.
• compositions for therapeutic use comprising at least one differentiation protein or a monoclonal antibody according to the invention conjugated to an active principle.
• fetal pancreatic extracts From fetuses in good condition, after therapeutic abortion, at 3 to 4 months of gestation, the pancreas which is rapidly immersed in an antiprotease solution containing aminocaproic acid (0.4 %) and aprotinin (40 units of Kallikreine / ml), for example aprotinin Trasylol ⁇ sold by Sigma.
• the pancreas at a rate of 100 mg / ml of antiprotease solution, is ground as soon as possible after removal with a Ultraturax mill for a few minutes in the cold.
• the extract is centrifuged at 20,000 rpm for 1/2 hour, the supernatant is aliquoted and stored at -80 ° C.
• pancreas extract is injected into a rabbit according to the following protocol:.
• Day 1 1 ml of extract + 1 ml of complete Freund's adjuvant in intradermal injections on several points on the back. .
• Day 24 Same operation as Day 1.
• Day 44 Test bleed n ° 1..
• Day 54 Re-immunization as Days 1 and 24.. Day 78; Test bleed # 2.
• Day 150 Intravenous injection without adjuvant (clear centrifuged solution).
• Day 156 Test bleed n ° 4.
• Day 186 New intravenous injection.
• the serum is then decomplemented, by bringing it to a water bath at 56 ⁇ C for approximately 30 minutes, then absorbed by extracts of normal adult pancreas, normal serum and red blood cells A, 0, B.
• Rinse with P.B.S. Incubate for 1 hour at 4 ° C in a saturated protein solution (2% egg white ovalbumin solution, in distilled water, marketed by Sigma) to block the binding sites of the membrane not occupied by l 'antigen. . Rinse with P.B.S. Incubate with antiserum.
• Organ sections fixed either with ethanol, formaldehyde or Bouin's liquid, are included in the paraffin. Deparaffinize, hydrate and inhibit endogenous peroxidase.
• the SDS-PAGE technique is used in 10% acrylamide gels in a Tris-glycine pH 8.3 buffer containing 0.1% SDS (Laenmli UK cleavage and structural proteins during assembly of head of the bacteriophage Nature (Lond. 227: 680-5 (1970)).
• nitrocellulose membranes are transferred to a Biorad transfer cell at 4 ° C. at 100 mA at constant current for 16 h at pH 8.3 (Towbin).
• the tasks are revealed as in the NIF technique.
• the adult pancreas were not colored.

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• 1984
  • 1984-10-01 FR FR8415081A patent/FR2571146B1/fr not_active Expired
• 1985
  • 1985-09-30 EP EP85401902A patent/EP0180496B1/de not_active Expired
  • 1985-09-30 US US06/878,838 patent/US4843019A/en not_active Expired - Fee Related
  • 1985-09-30 DE DE8585401902T patent/DE3570677D1/de not_active Expired
  • 1985-09-30 WO PCT/FR1985/000268 patent/WO1986002081A1/fr unknown
  • 1985-09-30 AT AT85401902T patent/ATE43610T1/de not_active IP Right Cessation
  • 1985-09-30 EP EP85904826A patent/EP0197974A1/de not_active Withdrawn
  • 1985-09-30 JP JP60504378A patent/JPS62500304A/ja active Pending
• 1986
  • 1986-05-30 OA OA58868A patent/OA08334A/xx unknown

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