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
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
  • C07K16/245—IL-1
• • 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
  • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6845—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1021—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against cytokines, e.g. growth factors, VEGF, TNF, lymphokines or interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2123/00—Preparations for testing in vivo
• • 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 anti-interleukin monoclonal antibodies, and in particular anti-interleukin 1 ⁇ and 1 ⁇ antibodies, to their production process, to anti-interleukin 1 ⁇ and 1 ⁇ / acetylcholinesterase monoclonal antibody conjugates, to enzymo-immunometric tests (EIA) for detecting and quantifying interleukins 1a and 1 ⁇ using said antibodies and / or conjugates and for various other applications of said antibodies and / or conjugates, in particular for therapeutic applications.
• EIA enzymo-immunometric tests
• Lymphokines are powerful pharmacological agents which result from the interaction between lymphocytes and the specific antigen which sensitizes them; they can modify, induce or suppress the functions of many cell types; they also play an essential role during inflammatory reactions and in the phenomena of bone resorption, fibrosis and chemotaxis. In addition, they are involved in the control of hematopoiesis and the regulation of immune responses. Lymphokines are produced by T cells, however it is known that some of them such as interleukin-1 can be produced by non-leukocyte cells.
• Interleukin-1 (cf. IMMUNOLOGY, Jeanêt BACH, Ch. XIII, pages 425-426, 3rd Edition 1986) is a mediator which constitutes a biological signal for the presentation of the antigen associated with the histotope, which marks the monocyte-T lymphocyte cooperation.
• the activity of IL-1 is traditionally measured in a proliferation test of mouse thymocytes C3H / HeJ (resistant to LPS) in the presence of PHA (I ⁇ g / ml). In the absence of IL-1, the thymocytes stimulated by the PHA are blocked in the Go / G1 phase. They can however divide if interleukin-2 is added, which suggests that the main action of IL-1 is the induction of interleukin-2 synthesis.
• Human IL-1 is a glycoprotein with a molecular weight estimated at 18 kDa, with a major contaminant at 15 kDa.
• two peptide fractions of molecular weights 2 and 4 kDa which exhibit the biological activity of IL-1 were isolated from normal urine.
• IL-1 appears to be produced by a wide variety of cell types excluding T cells, such as monocytes, macrophages, keratinocytes, astrocytes, melanocytes, mesangial cells, B lymphoblasts.
• T cells such as monocytes, macrophages, keratinocytes, astrocytes, melanocytes, mesangial cells, B lymphoblasts.
• IL-1 participates in the activation of B cells, stimulates the proliferation of fibroblasts and synoviocytes, raises the body temperature by acting on thermoregulatory centers and induces the production of proteins of inflammation by hepatocytes.
• An IL-1 gene encoding a precursor of 270 amino acids has recently been cloned. Plasmids containing this gene induce the production of a carboxy-terminal peptide of 156 amino acids, having the activities of IL-1. While in the aforementioned reference, the hypothesis of a family of molecules of neighboring structure was put forward, DINARELLO [J. CLIN. IMMUNOL.
• IL-1 represents an important family of biologically active proteins derived from mononuclear cells, which are involved in inflammatory reactions and in immune responses.
• Two distinct species of human IL-1 have been identified: IL-1 ⁇ and IL-1 ⁇ [MARCH et al., NATURE (1985), 315, pages 641-647]; these two species have the same molecular weight, similar biological effects and the same receptors on target cells [WOOD et al., J. IMMUNOL. (1985),
• Patent Applications also describe anti-IL-1 ⁇ and / or anti-IL-1 ⁇ antibodies.
• the OTSUKA PHARMA-CEUTICAL 267 611 European Patent Application describes monoclonal anti-IL-1 ⁇ antibodies and monoclonal anti-IL-1 ⁇ antibodies.
• the affinity constant of these antibodies is of the order of 3.6 ⁇ 10 -8 M / l.
• the European Patent Application IMMUNEX 245,052 describes more particularly a method of detecting the inflammation during which IL-1 intervenes, comprising (a) the reaction of a sample of biological fluid possibly containing IL- 1 with a first antibody specific for a first antigenic site characteristic of IL-1 and (b) detecting the reaction between the first antibody and IL-1.
• the antibodies more particularly described in this IMMUNEX Application are a monoclonal antibody which reacts specifically with IL-1 ⁇ (monoclonal antibody called 15A4) and a monoclonal antibody which reacts specifically with IL-1 ⁇ (monoclonal antibody called 7B4).
• the antibodies described in this IMMUNEX Application are a monoclonal antibody which reacts specifically with IL-1 ⁇ (monoclonal antibody called 15A4) and a monoclonal antibody which reacts specifically with IL-1 ⁇ (monoclonal antibody called 7B4).
• the antibodies described in this IMMUNEX Application are a monoclonal antibody which reacts specifically with
• European Patent Application DAINIPPON 220 063 describes an antibody directed against IL-1, said antibody being used in particular in the context of a competitive method or an immunometric method.
• This antibody only makes it possible, within the framework of these assays, to obtain sensitivities of the order of 0.1 ng / ml for the competitive method and of the order of 0.2 ng / ml for the immunometric method.
• French Patent No. 83 13389 in the name of the Applicant, has proposed conjugates in which an antibody, an antigen or a hapten is covalently linked to acetylcholinesterase, for the production of enzymoimmunoassays, thanks to the properties of the enzyme, carrying out enzyme assays of very small quantities, of the order of a nanogram.
• the anti-PS1 monoclonal antibodies present in the supernatants of hybridoma cultures bind to the solid phase, which results in the indirect immobilization of the corresponding biotinylated component of the PSI; - This biotinylated antigen strongly binds avidin, which itself binds the labeled AChE to biotin; - the activity of the fixed AChE is measured by the colorimetric method of ELLMAN.
• the Authors have shown that the AChE system significantly improves the detection limits of monoclonal antibodies. This screening method applies perfectly to the quantitative determination of different PS1 polypeptides by competition.
• the present invention has therefore given itself the aim of providing means suitable for enabling the performance of highly specific and sensitive enzymo-immunometric tests, of detection and quantification of IL-1 ⁇ and IL-1 ⁇ , these means also being capable of therapeutic applications and in vivo diagnostic applications.
• the subject of the present invention is monoclonal antibodies specific for IL-1 ⁇ and 1 ⁇ , which result from the immunization of mammals, in particular rodents, and more particularly of mice, with IL-1 ⁇ or -1 ⁇ as appropriate, which specifically recognize natural or recombinant IL-1 ⁇ or natural or recombinant IL-1 ⁇ , which show practically no cross-reaction between the two IL-1, characterized in that said monoclonal antibodies have a dissociation constant of less than 5.10 - 10 , in that they consist of several groups which recognize distinct regions of IL-1 ⁇ or -1 ⁇ , in that the antibodies of certain groups are capable of binding in a compatible manner with all the antibodies of the other groups, to know : - anti-IL-1 ⁇ antibodies are made up of three groups (A, B and C) which recognize three distinct regions of interleukin-1 ⁇ , all the antibodies in a group being capable of binding in a compatible manner with all those of the other two groups but not being compatible with another antibody of the same group;
• - anti-IL-1 ⁇ antibodies are made up of four groups (A, B, C and D), the first two (A and B) having a behavior similar to that of anti-IL-1 ⁇ antibodies, including group antibodies C are compatible with a single antibody from each of the first two groups and whose antibodies from the fourth group (D) are incompatible with any of the antibodies from the first three groups, and do not bind to the recombinant native IL-1 ⁇ , recognize a modified interleukin-1 ⁇ , and react with an IL-1 ⁇ -AChE conjugate;
• anti-IL-1 ⁇ and anti-IL-1 ⁇ antibodies recognize the IL-1 ⁇ or 1 ⁇ / acetylcholinesterase (AChE) conjugates.
• the monoclonal antibodies specific for interleukins-1 ⁇ and 1 ⁇ are obtained from mammals such as, in particular, rodents, and more particularly mice, which are immunized by appropriate injections of interleukin- 1 ⁇ or -1 ⁇ , then spleen cells of immunized mammals are fused, according to an appropriate technique, with mammalian myeloma cells of the same species, the resulting hybridomas are cultured and those in the supernatants of which were detected anti-interleukin-1 ⁇ or 1 ⁇ antibodies, as the case may be, are subcloned to obtain cell lines secreting anti-IL-1 ⁇ monoclonal antibodies or anti-IL-1 ⁇ , according to the interleukihe against which they were directed.
• the interleukin-1 ⁇ or -1 ⁇ used to immunize the abovementioned mammals can be both a purified natural interleukin and a recombinant interleukin.
• hybridomas used to obtain the anti-IL-1 ⁇ and anti-IL-1 ⁇ monoclonal antibodies in accordance with the invention were the subject of a deposit dated December 8, 1988 with the NATIONAL COLLECTION OF CULTURES OF MICROORGANISMS (CNCM) held by the INSTITUT PASTEUR.
• the deposited hybridomas are identified by the following numbers: I-823, I-824, I-825, I-826, I- 827, 1-828, 1-829.
• the present invention also relates to conjugates of acetylcholinesterase and of anti-IL-1 ⁇ or -1 ⁇ monoclonal antibodies or of fragments of these antibodies, in particular of Fab 'fragments.
• the present invention further relates to a process for producing Fab 'fragments of anti-interleukin-1 ⁇ or 1 ⁇ monoclonal antibodies in accordance with the present invention.
• said monoclonal antibodies are treated with pepsin in an acid medium, to obtain fragments F (ab ') 2 which are isolated by chromatography on a molecular sieve, then these fragments F (ab') 2 are subjected to a controlled reduction process, by an appropriate reducing agent such as, in particular, ⁇ -mercaptoethylamine ( ⁇ MEA), to provide a Fab 'fragment which is purified by passage through a column chromatography column.
• an appropriate reducing agent such as, in particular, ⁇ -mercaptoethylamine ( ⁇ MEA)
• the present invention also relates to a method for producing AChE conjugates and anti-IL-1 ⁇ or anti-IL-1 ⁇ monoclonal antibodies and AChE conjugates and fragments of said monoclonal antibodies, in particular Fab 'fragments , which is characterized in that at least one incorporated or natural thiol group- ment present in the antibody or one of its fragments, is coupled to at least one maleimido group incorporated in AChE, via an appropriate coupling agent such as, in particular, an ester of N-succinimide such as N-succinimidyl-4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC).
• an appropriate coupling agent such as, in particular, an ester of N-succinimide such as N-succinimidyl-4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC).
• the present invention further relates to enzyme immunoassays by competition for the detection of Il-1 ⁇ and IL-1 ⁇ in cell culture supernatants and in biological fluids, characterized in that a monoclonal antibody anti-IL-1 ⁇ or anti-IL-1 ⁇ according to the invention, used as first antibody, and IgG, carried by an appropriate solid support, used as second antibody, are brought into contact with conjugates of IL-1 -AChE, which play the role of tracers, and of interleukin-1-1 ⁇ or -1 ⁇ , after which the AChE activity is revealed by any appropriate means.
• the present invention also relates to highly specific and sensitive enzymo-immunometric tests for the detection of Il-1 ⁇ and IL-1 ⁇ in cell culture supernatants and biological fluids, characterized in that antibodies monoclonal (mAb) anti-IL-1 ⁇ and / or anti-IL-1 ⁇ according to the invention and carried by an appropriate support, are brought into contact with IL-1 ⁇ or -1 ⁇ and with the Acm-AChE conjugate , after which the AChE activity is revealed by any appropriate means.
• mAb monoclonal
• IL-1 ⁇ or -1 ⁇ is added to the antibody simultaneously with the conjugate Acm-AChE and the two monoclonal antibodies react simultaneously with IL-1 ⁇ or - 1 ⁇ .
• the IL-1 ⁇ or -1 ⁇ is first introduced and reacts with the antibody, then the Acm-AChE conjugate is then introduced and the activity
• AChE is revealed by any appropriate means.
• the suitable supports are in particular microtiter plates or supports in the form of particles allowing the fixing of said antibodies.
• the subject of the present invention is, moreover, a kit for carrying out immunoenzymatic tests by competition in accordance with the invention, characterized in that it comprises:
• At least one bottle containing appropriate doses of anti-IL-1 ⁇ monoclonal antibody and / or anti-IL-1 ⁇ monoclonal antibody;
• the present invention also relates to a kit for carrying out the enzymo-immunometric tests in accordance with the invention, characterized in that it comprises:
• the present invention further relates to agents for therapeutic purposes, characterized in that they consist of, or comprise as active constituents, anti-IL-1 ⁇ monoclonal antibodies and / or anti-IL antibodies -1 ⁇ and / or their fragments, co to the invention, alone or conjugated or hybrid or associated with other substances.
• a particularly advantageous therapeutic application of said anti-IL-1 ⁇ and anti-IL-1 ⁇ monoclonal antibodies is their activity of inhibiting IL-1.
• the present invention also relates to diagnostic agents capable of being used for in vivo diagnosis, characterized in that they comprise anti-IL-1 ⁇ antibodies and / or anti-IL-1 ⁇ antibodies or their fragments , associated with an antigen and / or a hapten and / or another antibody or antibody fragment, and / or labeled with stable or radioactive labels.
• FIG. 4 the in vitro release of IL-1 ⁇ ( ⁇ , ⁇ ) and IL-1 ⁇ ( ⁇ , ) established by specific EIAs in the supernatants of unstimulated human monocytes, lymphocytes and neutrophils ( ⁇ , ⁇ ) and stimulated by LPS ( ⁇ , ), purified by centrifugal elutriation.
• EXAMPLE 1 Implementation of the tests in accordance with the invention; preparation of reagents (monoclonal antibodies and conjugates) and reagent selection parameters.
• the acetylcholinesterase (AChE) obtained from the electric eel Electrophorus electricus was purified by affinity chromatography by the method described by MASSOULIE and BON [Eur. J. BIOCHEM (1976), 68, pages 531-539].
• the tetrameric form of the enzyme, or G4 form has been used to label interleukins and antibodies.
• AChE activity was measured using the colorimetric method of ELLMAN et al. [BIOCHEM. PHARM. (1961), 7, pages 88-95].
• An ELLMAN unit is defined as the quantity of enzyme producing an increase in absorbance of 1 DO at 25 ° C, for 1 minute, in 1 ml of medium and for an optical step length of 1 cm; it corresponds to approximately 8 ng of enzyme and to 7.32 enzymatic units (one enzymatic unit corresponds to the quantity of enzyme which hydrolyzes 1 ⁇ mol of acetylcholine at 25 ° C. for 1 minute).
• AChE concentrations were determined enzymatically using a catalytic constant of 4.4 x 10 7 moles h -1 per site and a molecular mass of 80 kDa for the catalytic subunit.
• a detection limit of 1.8 attomole (10 -18 mole) of enzyme can be calculated for the G4 form (i.e. the amount of AChE which produces an increase in absorbance of 0.01 DO for 1 hour, in 20 ⁇ l of ELLMAN medium, for a step length of 0.5 cm.
• Anti-interleukin-1 ⁇ and 1- ⁇ antibodies were produced in Biozzi High Responder (HR) mice using the following immunization process: on day 0, 15 ⁇ g of recombinant interleukin la or 1 ⁇ emulsified in Freund's complete adjuvant was injected into the pad of one leg. The mice were treated with aspirin (0.1 mg / day) to avoid side effects such as hyperthermia. A first booster injection (in the pad of a paw) was administered on day 21 and the mice were bled a week later. The presence of murine anti-interleukin antibodies in the corresponding sera was checked by testing their capacity to fix interleukin-1-AChE conjugates.
• HR Biozzi High Responder
• mice received an intravenous booster injection (7 ⁇ g of interleukin) 3 and 2 days before the fusion.
• Corresponding mouse spleen cells were fused with NSI mouse myeloma cells, as described in the article by GRASSI et al. cited in the preamble.
• the interleukins 1a and 1 ⁇ were covalently coupled to AChE via a heterobifunctional reagent, namely N-succinimidyl-4- (N- maleimido-methyl) cyclohexane-1-carboxylate (SMCC) using a known technique for labeling bovine acid growth factor (aFGF) and rat pro-lactin.
• SMCC N-succinimidyl-4- (N- maleimido-methyl) cyclohexane-1-carboxylate
• aFGF bovine acid growth factor
• rat pro-lactin bovine acid growth factor
• This method involves the reaction of a thiol group (previously introduced into interleukins) with a maleimido group incorporated into the enzyme after the reaction with SMCC.
• the interleukins were thiolated by reaction of their primary amine groups with N-succinimidyl-S-acetyl-thioacetate (SATA)
• AChE Maleimido groups were introduced into AChE (form G4) by reaction with SMCC; if not used immediately, the AChE-SMCC preparation can be frozen at -80 ° C and stored without losing its reactive properties, for at least several weeks.
• the thiolated interleukins were coupled to the preparation of AChE-SMCC (form G4) by mixing the two reagents immediately after they were isolated by molecular sieve chromatography or as soon as they were thawed. At this stage, a molecular ratio (SH-interleukin / G4-SMCC) of 50/1 was used.
• Monoclonal antibodies have been purified from ascites fluids by precipitation with caprylic acid and ammonium sulfate, according to a technique described in the prior art. The purity of the preparation was checked by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Two different labeling methods were used to couple purified mAbs to AChE.
• Biotin has been covalently linked to mAbs as defined above by reaction of an activated biotin N-hydroxysuccinimide ester (IBF, France) with the primary amino groups of the antibodies.
• the activated ester was dissolved in anhydrous dimethylformamide and added to an alkaline solution of the antibody to be labeled: 40 ⁇ l of a 5 mg / ml solution of biotin ester in DMF, to 1 mg of was added.
• antibody dissolved in 2 ml of 0.1M borate buffer, pH 8.5. After 30 minutes at room temperature, 2 ml of EIA buffer (which will be defined later) was added. This preparation was used to determine the "binding compatibility" of the various mAbs without undergoing further purifications. It was stored frozen at -20oC.
• Acm Fab 'fragments were covalently coupled to AChE via the SMCC using a technique derived from that described by ISHIKAWA et al. [J. IMMUNOASSAY (1983) 4, pages 209-327] for labeling antibody fragments with other enzymes.
• the principle of the method used is very similar to that used to label the interleukins, which has been described above, except that, in the present case, the thiol groups involved in the coupling reaction are naturally present in the Fab 'fragments obtained by reduction of the corresponding F (ab') 2 fragment.
• Fragments F (ab ′) 2 were obtained by treatment of Acm with pepsin in an acid medium (acetate buffer pH 4.3). They were isolated from the crude preparation treated with pepsin, by chromatography on molecular sieve on a column (30 x 1.5 cm) of 0.5 m Biogel equilibrated in 0.1 M phosphate buffer, pH 6, containing 5.10 -3 M EDTA. The purity of fraction F (ab ') 2 was verified by polyacrylamide gel electrophoresis under non-reducing denaturing conditions. These measurements showed that more than 80% of the total protein content was composed of fragments of F (ab ') 2.
• Fragments F (ab ') 2 were reduced in the presence of 0.1M ⁇ -mercaptoethylamine ( ⁇ MEA) at 37 ° C for 1 hour.
• ⁇ MEA ⁇ -mercaptoethylamine
• the excess ⁇ MEA was removed by chromatography on a molecular sieve on a column (30 x 1.5 cm) of Sephadex G25, as described above for the thiolated interleukins.
• the concentration of Fab 'fragments was measured by UV spectrometry using the extinction coefficient at 280 nm, 1.48 g - 1 . L cm -1 and as molecular weight 46000.
• the content of thiol groups in the Fab 'preparation was determined by reaction with DTNB, as for the thiolated interleukins. Depending on the mAb used, values of between 1 and 4 SH groups have been found per molecule of Fab '.
• AChE form G4
• SMCC cyclopentadiene carboxylate
• the coupling of the enzyme with Fab 'fragments was carried out by mixing the AChE-SMCC preparation (immediately after it was isolated by chromatography on molecular sieve or thawed) with an excess of Fab 'fragments. Usually a molar ratio of 50 is used at this stage (i.e. thiol groups of Fab '/ G4-SMCC). After reaction for 3 hours at 30 ° C, the G4-Fab 'conjugates were isolated by chromatography on molecular sieve on a 0.5 m Biogel column.
• the conjugate was eluted in the form of a single homogeneous peak, the corresponding fraction was collected and stored in the frozen state at -20oC. No significant loss of enzymatic activity was observed during the entire coupling process. The stability of the conjugates proved to be excellent because they could be stored either in the frozen state at -20o C, or in the lyophilized state, or in the liquid state at + 4oC, without losing their enzymatic or immunological properties. . 5. SCREENING OF CULTURE SURNANTANTS:
• the presence of anti-interleukin-1 antibodies in supernatants of hybridoma cultures was detected using an enzyme immunoassay similar to that described above.
• the culture supernatants were incubated in microtiter plates coated with a second anti-mouse IgG antibody, together with conjugates of interleukin 1-AChE.
• the anti-interleukin 1 mAbs optionally present in the supernatants bind simultaneously to the IL-1-AChE conjugate and to the solid phase, which results in the indirect immobilization of the AChE activity on the plates.
• the presence of AChE on the solid phase was further revealed (after a washing step) by adding the substrate and measuring by colorimetry.
• the preparation and characteristics of the second solid phase antibody are well known; 50 ⁇ l of each supernatant present on the microtitration plates comprising 96 wells, were transferred under sterile conditions into microtitration plates of the same dimensions, coated with the second antibody. 50 ⁇ l of conjugate ford interleukin 1-AChE (1 ELLMAN unit / ml) dissolved in EIA buffer were then added and the mixture was allowed to react overnight at + 4 ° C. At the end of this first reaction period, the plates were washed thoroughly before adding 200 ⁇ l of ELLMAN reagent to each well.
• the binding compatibility of two different mAbs on a single molecule of Interleukin-1 was determined in an immunometric test in which one of the mAbs was immobilized on a solid phase while the other was labeled with biotin molecules .
• These tests were carried out as follows: all the dilutions were carried out in EIA buffer and 100 ⁇ l of a solution at 100 ng / ml of recombinant interleukin-1 ⁇ or 1 ⁇ were added to wells of microtitration plates coated with one of the Acm. After one hour of reaction at room temperature with stirring, 100 ⁇ l of a 10 ⁇ g / ml solution of another biotin-labeled mAb were added.
• Monoclonal antibodies from culture supernatants or ascites fluids were first tested in a competitive immunoassay using IL-1-AChE conjugates as tracers.
• immunometric tests were developed involving the simultaneous use of anti-interleukin mAbs, either coating a solid phase or marked with AChE.
• All the reagents used in the immunoassay were diluted in the EIA buffer mentioned above, the composition of which is as follows: 0.1 M phosphate, pH 7.6, containing 0.4 M NaCl, 10 -3 M EDTA, 0.1% BSA and 0.01% sodium azide. All the concentrations mentioned in the immunoassays refer to the concentration of the reagents in the "initial volume" (50 ⁇ l for the competition test, 100 ⁇ l for the immunometric test) before mixing with the other reagents.
• the working dilutions of the mAbs were determined beforehand by carrying out antibody dilution curves from culture supernatants or ascites fluids.
• Immunometric tests were carried out in microtiter plates comprising 96 wells, coated with one of the monoclonal antibodies. The coating was carried out exactly as described above for the second antibody in solid phase. Briefly, the wells of the microtitration plates were filled with 200 ⁇ l of a 10 ⁇ g / ml solution of mAb dissolved in 5 ⁇ 10 -2 M phosphate buffer, 7.4 mH. After allowing the reaction to occur overnight at room temperature, the plates were washed thoroughly with 10 -2 phosphate buffer, pH 7.4 containing TWEEN 20, 0.05%. The solid phase was then saturated by adding 300 ⁇ l of EIA buffer to each well for at least 4 hours at room temperature. Plates were stored at 4 ° C in this saturation buffer until that they be used. When stored under these conditions, no changes in their fixing properties were observed for at least a period of four months. The immunometric tests were carried out using two different procedures.
• the first protocol (called “Simultaneous Protocol") consisted in adding 100 ⁇ l of interleukin-1 in solution (introduced in the form of a recombinant standard or a sample) into wells of microtitration plates, together with 100 ⁇ l of Acm-AChE conjugates (usually at a concentration of 10 ELLMAN units / ml).
• the two antibodies (the solid phase antibody and the antibody labeled with AChE) react simultaneously with interleukin-1.
• interleukin-1 is dissolved standard recombinant in the corresponding diluent, i.e. culture medium, plasma or serum.
• AChE fixed to the solid phase was determined by adding 200 ⁇ l of ELLMAN reagent.
• a second protocol (called “sequential protocol”) consists in reacting interleukin-1 and the Acm-AChE conjugate separately.
• first incubation period 200 ⁇ l of interleukin-1 (introduced in the form of a standard or sample) dissolved in EIA buffer or any other appropriate diluent (culture medium, plasma, etc.) .) react with the solid phase.
• EIA buffer or any other appropriate diluent (culture medium, plasma, etc.) .
• the plates are washed before adding 200 ⁇ l of Acm-AChE conjugate (usually at 10 ELLMANN units / ml) dissolved in EIA buffer.
• the plates are washed again and the AChE activity is revealed as described above.
• Standard curves "imprecision profiles" were established by performing all the measurements (non-specific fixation as well as standard measurements) eight times. For each dose, the precision was expressed in terms of coefficient of variation (CV%) and was plotted as a function of the dose logarithm.
• MDC minimum detectable concentration
• An immunoreactive substance detected by immunometric tests in biological media has been characterized by fractionating samples by molecular sieve chromatography using FPLC equipment and a Superose 12 column equilibrated with EIA buffer. Standard recombinant IL-1 and samples were injected in a volume of 500 ⁇ l and at a flow rate of 24 ml / hour and then 0.8 ml fractions were collected. At the end of the chromatography, 100 ⁇ l of each of the fractions was tested by implementing the IL-1 ⁇ and / or IL-1 ⁇ test as described above.
• Mononuclear leukocytes (lymphocytes and monocytes) and polymorphonuclear (neutrophils) from peripheral blood were obtained by centrifugation on FICOLLPAQUE (PHARMACIA FINE CHEMICALS) and were then subjected to countercurrent centrifugal elutriation to allow the production of lymphocytes, pure monocytes and neutrophils.
• FICOLLPAQUE PARMACIA FINE CHEMICALS
• the elutriation buffer consisted of a saturated PBS solution from Dulbecco supplemented with 0.25% BSA and 2 mM EDTA.
• the platelets were washed after half an hour of elutriation, at the injection rate.
• the fractionation of mononuclear leukocytes was carried out as follows: pure lymphocytes were obtained by increasing the flow rate from 8 to 20 ml / min.
• the mouse whose serum had the highest titer in the enzyme immunoassays was selected for fusion.
• the corresponding spleen cells were fused with NSI myeloma cells as described above.
• One week after the fusion approximately 500 wells presented hybridomas which were actively multiplying for the two interleukins.
• the presence of anti-interleukin mouse antibodies was verified in all the culture supernatants by testing their capacity to fix the corresponding IL-1-AChE conjugate. In a primary screening, 90 and 116 strongly positive culture supernatants were detected for IL-1 ⁇ and IL-1 ⁇ respectively.
• the corresponding hybridomas were subcloned by limiting dilutions in order to obtain a cell line secreting antibodies, stable, unique for each culture. Finally, 36 and 11 cell lines were respectively stabilized for IL-1 ⁇ and IL-1 ⁇ . All the clones were characterized by a number preceded by the letter ⁇ or ⁇ according to the interleukin against which they had been directed. They were cultured as ascites tumors and the monoclonal antibodies were purified from ascites fluid by precipitation with caprylic acid and ammonium sulfate. The isotypes were determined from ascites fluid by the OUCHTERLONY double diffusion technique. All of the mAbs were IgGs having a K light chain, with IgG 1 being by far the most common (see Tables I and II below).
• Example 1 The binding compatibility tests were carried out as described above in Example 1.
• the solid circles indicate couples for which a simultaneous binding of the mAbs immobilized in solid phase and of the labeled mAbs was observed.
• the mAbs with the same definition have been grouped into three categories (A to C). All mAbs are in the IgG 1 subclass except mAb 100 (group A) and mAbs 50 and 176 (group B) which are IgG 2 .
• Example 1 The binding compatibility tests were carried out as described above in Example 1. The solid circles indicate couples for which a simultaneous binding of the mAbs immobilized in solid phase and of the labeled mAbs was observed. To clarify the presentation, mAbs with the same type of fixation have been grouped into four categories (A to D). The IgG subclass of each mAb is mentioned in the last column. Culture supernatants as well as ascites fluids have been used in competitive enzyme immunoassays involving IL-1-AChE conjugates as tracers. In most cases (except for the ⁇ 26 and ⁇ 54 clones), standard curves could be established using recombinant IL-1 ⁇ and IL-1 ⁇ as standards.
• One of its essential aims is to develop immunometric tests at two sites for both IL-1 ⁇ and IL-1 ⁇ , so that it is necessary to determine which pairs of MAbs which could be attached simultaneously to each interleukin.
• These binding complementarity studies were carried out using immunometric tests in which one of the mAbs was immobilized on the solid phase while the other was labeled with biotin molecules.
• labeling with biotin was chosen because it is a completely simple which allows the marking of a large number of AMC
• Tables I and II above present the results of these additional tests for IL-1 ⁇ and IL-1 ⁇ respectively.
• IL-1 ⁇ we see that there are three groups of mAbs which recognize epitopes which are found in three different regions of the molecules of IL-1 ⁇ . These groups, which have been designated by A, B and C, contain 10, 25 and 1 monoclonal antibodies, respectively. All the mAbs of a group have a binding compatible with all the mAbs of the other two groups, while they do not bind simultaneously to each other or with mAbs of the same group. In the case of IL-1 ⁇ , the situation is less clear. In fact, there are four different groups.
• the mAbs which gave the best results were marked and the tests were continued. with them using the corresponding immobilized mAbs.
• the antibodies ⁇ 5, ⁇ 29, ⁇ 100, ⁇ 147 (group A), ⁇ 176, ⁇ 39 (group B), and ⁇ 101 (group C) were successively labeled and tested.
• FIG. 1 summarizes the main characteristics of the tests for detecting IL-1 ⁇ and IL-1 ⁇ in the case of an overnight incubation at 4 ° C. and d 'a simultaneous process.
• the two tests seem very sensitive because minimum detectable concentrations lower than 4 pg / ml could be calculated for each interleukin by using a period of 30 minutes for the enzymatic measurement. Increased sensitivity is observed when the enzymatic reaction (revelation step) lasts longer. In such a case, minimum detectable concentrations below 2 pg / ml can be obtained (see Table IV below).
• the identity between the substance detected in the culture supernatants (or the intracellular extracts of cells) and the recombinant IL-1 used to establish the standard curves is confirmed by three types of control experiments. The first is represented by the dilutions of samples in culture medium, which gave dilution curves parallel to the standard curves. This is shown in Table V below, which presents the results of two different tests relating to IL-1 ⁇ and IL-1 ⁇ , carried out at different dilutions. Similar results were obtained with all of the samples tested.
• Samples A and B were diluted as indicated in Table V and tested for IL- la and IL-1 ⁇ by EIA. Both the dilutions and the standard curve were made in culture medium (RPMI + 10% fetal calf serum). The raw data were multiplied by the dilution factor before being listed in the Table, in order to clearly show the parallelism between the standard curve and the dilution curves of the samples.
• Sample A supernatant of alveolar macrophages in a culture stimulated by TNF ⁇ .
• Sample B intracellular extract of alveolar macrophages in culture.
• the third experiment consisted in the analysis of culture supernatant after fractionation by chromatography on molecular sieves; this analysis shows that the immunoreactive substance is eluted in the form of a single homogeneous peak, as already indicated above, which corresponds exactly to the peak observed with the recombinant interleukins.
• FIG. 7 shows different chromatographic profiles obtained for supernatants of stimulated alveolar macrophages, both in the test for detecting IL-1 ⁇ and in the test for detecting IL-1 ⁇ . Similar results have been observed with other culture supernatants or with intracellular extracts. Taken as a whole, all these data clearly indicate that the two tests effectively allow a quantitative determination of interleukins 1 in these environments.
• EXAMPLE 4 Measurement of IL-1 ⁇ and iL-1 ⁇ in plasma and serum.
• JURKAT cells for example (cell line derived from a human T lymphoma) secrete IL-2 when they are stimulated by IL-1 ⁇ or IL-1 ⁇ in the presence of phytohemagglutin (PHA) ).
• Certain anti-IL-1 ⁇ or anti-IL-1 ⁇ antibodies are capable of inhibiting the effect of IL-1 either because they recognize the site involved in the binding with the receptor or because the formation of the IL-1 complex - antibody disrupts the biological process.
• the anti-IL-1 ⁇ or IL-1 ⁇ antibodies according to the invention (10 ⁇ g / ml) are incubated in the presence of IL-1 ⁇ or IL-1 ⁇ (10 ng / ml) for 18 hours at 4 ° C. .
• a control is carried out by incubating IL-1 ⁇ or IL-1 ⁇ (10 ng / ml) without antibodies under the same conditions.
• the concentration of IL-1 at the time of stimulation is 1 ng / ml.
• the concentration of IL-2 in cell culture supernatants is measured using an immunometric assay developed at SPI, the principle of which is identical to those developed for IL-1.
• results are expressed as a percentage of inhibition compared to two controls, one is carried out in the absence of IL-1 (PHA alone) and represents 100% inhibition, the other is carried out in the absence of antibody. and represents 0% inhibition.
• IL-1 ⁇ acts on cells via a membrane receptor with which it specifically binds.
• Monoclonal antibodies can inhibit the binding of IL-1 ⁇ to receptors if they recognize the part of the IL-1 ⁇ molecule involved in the complex. By inhibiting the binding with the receptor they also inhibit the biological effect.
• the iodine-labeled IL-1 is incubated for one hour at 4 ° C. with the antibodies at a concentration of
• the mixture is brought into contact with EL4 cells (murine Thymoma cell line) for 8 hours at + 4 ° C.
• the cells are then washed with an appropriate buffer and the fixed radioactivity is measured with a solid scintillation counter (multigramma LKB). The results are expressed as a percentage of inhibition relative to a control (100% binding) for which the IL-1 ⁇ labeled with iodine 125 has not been incubated with an antibody.
• EXAMPLE 6 Demonstration of the sensitivity of the monoclonal antibodies in accordance with the invention; comparative test with tests using the antibodies of the prior art.
• Example 2 above shows that in the optimal reactions of the tests in accordance with the invention, the minimum detectable concentrations are less than 2 pg / ml (see in particular Table IV).
• Example 1 the sensitivity of the tests was. characterized by the dose of IL-1 which induces a statistically significant reduction in the fixation observed in the absence of competitor (Bo), that is to say
• MEDGENIX immunoradiometric assay: anti-IL-1 ⁇ antibody labeled with iodine 125
• the curve is obtained as visible in FIG. 9, which includes the concentrations of IL-1 ⁇ on the abscissa in pg / ml and on the ordinate the radioactivity bound (in cpm) and which shows that the detection limit is of the order of 15 pg / ml.
• the sensitivity of the immunometric tests is calculated as the dose of IL-1, which corresponds to non-specific binding + 3 standard deviations (with a confidence rate of 99%). This therefore corresponds to a significantly lower sensitivity than that of the tests in accordance with the invention, as specified above, which is of the order of 2 pg / ml.

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