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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4726—Lectins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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 invention relates to compounds having lectin properties, and to their biological applications.
• sarcolectins are also found in a wide variety of normal or tumor tissues in vertebrates (primates, rodents, gallinaceous).
• Pronase R but also resistance to mild treatment, under certain conditions, with pepsin; resistance to thermal variations
• interferon interferon
• the SCLs inhibit the further synthesis of interferon-dependent proteins, for example the kmase protein and 2-5A synthetase.
• the restoration of the initial state depends on the doses of SCL and can be more or less complete.
• the invention is based on obtaining highly purified SCL preparations which have enabled to develop strategies leading to the isolation of cDNA clones coding for a 55 kD protein, the study of which has revealed unexpected biological properties.
• the 65kD protein as a molecule comprising the biological properties of SCL.
• the major band in the 65kD region was indeed retained as carrying all the biological properties: the 55kD band is not constantly observed and moreover appears minor in all cases.
• the 65kD band corresponds to an artefact which results from the binding of a few molecules of SCL to albumin, but that the molecule having sarcolectm-like properties is actually the 55kD protein which contains all the genetic information responsible for the biological expression of the molecule.
• the object of the invention is therefore to provide various products relating to the 55 kD SCL, namely in particular proteins, polypeptides or their fragments, DNA sequences coding for these proteins or polypeptides, or their fragments, or on the contrary. inhibiting their expression, and antibodies directed against these proteins, polypeptides, or their fragments.
• sarcolectm or SCL, as used in the rest of the description, will denote either the proteins, polypeptides and fragments of these compounds, or their derivatives, as soon as they have lectin properties as defined according to the invention. It also relates to procedures for obtaining these various products.
• the invention further relates to the biological applications of these products.
• the nucleotide sequences according to the invention are sequences isolated from their natural environment and are characterized in that they comprise at least part of the sequence SEQ ID No. 1, one or more nucleotides being modified if necessary, it being understood that these sequences are capable of coding for sarcolectins, that is to say proteins, polypeptides, or fragments of these compounds, or alternatively derivatives, having lectinic properties.
• sequence SEQ ID No. 1 is given at the end of the description, with the other sequences mentioned below, in the document called "List of sequences”.
• lectinic properties is meant the capacity of SCLs to agglutinate normal or transformed cells, their stimulating effect on cell growth and their inhibitory effect on the anti-viral effect induced by interferon, under the conditions described in (8). .
• sequences implemented according to conventional recombinant DNA techniques, are capable of coding for proteins or polypeptides, or their fragments, exhibiting lectmic activity, comprising at least one chain of animated acids as indicated on SEQ ID N ° 1, in which one or more amino acids are, if necessary, modified.
• sequences are further characterized in that they are capable of hybridizing with at least one fragment of SEQ ID N ° 1 carrying at least part of the genetic information for a sarcolectm. This hybridization can be carried out under stringent conditions, but also under relaxed conditions as described in (10).
• the invention relates in particular to the nucleotide sequence corresponding to the open reading frame going from position 62 to position 1469 in SEQ ID NO:
• the domains of the 5 ′ ends and / or 3 ′ ends are especially preferred, since they contain the genetic information for the fragments possessing said lectinic properties.
• the sequence of approximately 405 bp from position 62 to 467 in SEQ ID NO: 1, as shown in SEQ ID NO: 2, is most particularly preferred.
• These 5 'and 3' domains are characterized in that they contain a high proportion of phosphorylatable amino acids, such as serine, threonme and tyrosme.
• sequences mentioned in the above can be genomic or genomic-type sequences, certain sequences of nucleotides can be separated by mtrons which will be excised to lead to the expression of mature SCLs.
• sequences derived from SEQ ID No. 1 are also targeted by the invention.
• These derived sequences are obtained by modification, substitution, alteration, mutation or genetic and / or chemical deletion of one or more nucleotides of SEQ ID No 1 or of a fragment of SEQ ID No 1, it being understood that they have genetic information for coding for an SCL conserving at least in part the lectmic activity presented by the polypeptide coded by SEQ ID No. 1, this activity being if necessary increased.
• the invention also relates to expression vectors containing at least one of the nucleotide sequences defined above, subjected to the control of an appropriate promoter. It targets in particular the vectors comprising all or part of SEQ ID N c 1 or SEQ ID No 2, or their antisense sequences.
• Host cells transfected with these vectors which include the expressed recombinant proteins are also part of the invention.
• the cells used for the transfections are, conventionally, eukaryotic or prokaryotic cells or even plant cells.
• the invention also relates, as new chemicals, to SCLs exhibiting lectinic properties as defined above.
• the invention thus relates to proteins or polypeptides, or their fragments, or their derivatives, characterized in that they have lectmic activity and comprise at least part of a sequence of amino acids in the sequence coded by l one of the nucleotide sequences defined above, one or more amino acids being modified if necessary, provided that this modification does not alter the lectinic properties of proteins, polypeptides, or their fragments, or their derivatives.
• the invention relates in particular to SCLs having a chain of amino acids in SEQ ID No. 3 or in
• the SCL corresponding to the open reading frame in the sequence SEQ ID No. 1 comprises 469 amino acids and has a molecular weight evaluated at 55 kD.
• the sequences of the 5 'and / or 3' regions of SEQ ID No. 1 comprises 469 amino acids and has a molecular weight evaluated at 55 kD.
• peptide sequences correspond to peptides having a structure corresponding to that of the sequence SEQ ID N ° 4, going from position 41 to 55 in SEQ ID N ° 1 or N ° 4.
• peptides having the sequence SEQ ID No. 5 or peptides derived from this sequence are characterized in that they are recognized by monoclonal antibodies capable of reacting with SEQ ID No. 1, by their own antibodies, but that they do not react with antibodies directed against the peptide fragment 81 to 95 in SEQ ID No. 1.
• Other preferred peptide sequences correspond to peptides of sequence SEQ ID No 6, having a structure corresponding to that of the sequence going from position 81 to 95 in SEQ ID No 1.
• derived peptides the sequences differing by one or more amino acids (in particular by modification, substitution, deletion) but recognized, by the same antibodies as the native sequences.
• the peptides SEQ ID N ° 5 and SEQ ID N ° 6, and their derivatives are capable, when fixed on a sensitive cell in vitro, of inhibiting the binding of the complete molecule and of hindering expression of its functions, the effects being less pronounced with the peptide SEQ ID No. 6 or its derivatives.
• the SCLs of the invention are further characterized in that they are as obtained by expression, in an appropriate host cell, according to recombinant DNA techniques, of an expression vector containing a DNA sequence as defined above, recovery of the expressed SCL and purification.
• SCLs of the invention are fragments or derivatives obtained by modification of the sequences SEQ
• modification is understood to mean any mutation, deletion, substitution and / or addition of one or more amino acids, obtained directly at the level of the amino acid chain, or indirectly by modifying the coding nucleotide sequence.
• Still other SCLs are as obtained by purification from tissue extracts.
• the invention relates in particular to purified SCLs as obtained from tissue extracts, by operating as follows:
• tissue extract containing lectins with pepsin in a gentle manner or at an acid pH, under conditions making it possible to eliminate at least the major part of the contaminating proteins, especially albumin, while retaining the activity lectmics,
• Highly purified SCLs are characterized in that they appear to be almost totally devoid of albumin. They give a unique peak in HPLC and show, after chromatography on SDS gel and denaturation, three characteristic bands formed of proteins of size estimated at 65 kD, 55 kD and ⁇ 14 kD.
• the invention relates especially to SCL characterized by a molecular weight of approximately 55 kD, as estimated in SDS-PAGE by comparison with polypeptides of defined molecular weight.
• the invention makes it possible on the one hand to characterize the molecule, on the other hand to obtain antibodies which are sufficiently pure to characterize the antigenic structure of SCLs.
• the invention also relates to methods for obtaining the nucleotide sequences and the SCLs defined above.
• nucleotide sequences it is possible to operate, according to conventional techniques, synthetically.
• a cDNA library is screened using specific probes, such as antibodies directed against the purified protein of 55 kD mentioned above and described in more detail in the examples, or of mRNA.
• the sequences sought are isolated, if necessary modified as desired, according to the applications envisaged, and subjected to one or more purification treatments.
• these sequences are advantageously introduced into an appropriate expression vector, under the control of a promoter, for the purpose of transfection of a host cell.
• the synthesis of the latter is obtained, and, after lysis of the cells or simply after excretion, we it is recovered, in recombinant form, and it is subjected to at least one purification step.
• prokaryotic systems such as bacteria, or eukaryotes, such as insect cells (Baculovirus system) or even yeasts, are advantageously used, as are commercially available.
• Animal cells such as hamster CHO or primate transfected with the appropriate gene can also be used.
• the SCLs of the invention can also be obtained by synthesis.
• the methods described above allow the purification of SCL molecules while retaining their biological properties.
• the invention relates in particular to a process for obtaining SCL of high degree of purity, from tissue extracts, characterized in that it comprises the steps of treatment of the tissue extract containing lectins with pepsin, gently, or at acidic pH, followed by chromatography under conditions making it possible to remove at least the majority of the contaminating proteins, in particular albumin, while retaining lectmic activity. These chromatography steps include - passing the pre-treated tissue extract over Sephacryl S-200,
• This succession of steps provides a technical solution to the problem of the separation of the contaminating albumin and makes it possible to eliminate it in spite of the size close to that of SCL, of certain physicochemical properties close to those of SCL, as well as possible interactions between albumin and SCL.
• the step of processing the tissue extract is carried out using pepsin, operating under controlled conditions.
• the respective concentrations are of the order of 4 to 8 o, preferably approximately 6 ° for the tissue extract and 0.5 to 2 mg / ml, preferably approximately 1 mg / ml for a pepsin having an activity of the order of 2500 to 2700 units / mg.
• Conditions for processing the tissue extract which have proved to be advantageous correspond to an incubation of the reactive mixture at approximately 37 ° C., pH 2, for approximately 1 h 30 to 2 h 30, in particular for 2 h.
• the enzyme activity is then stopped by increasing the pH to a value close to neutrality.
• this extract is previously centrifuged, and the supernatant is subjected to filtration. on a Sephacryl S-200 gel.
• the active fractions are recovered, eluting with a buffer solution such as PBS, at a rate of 15 to 25 ml / h.
• Elution is carried out with the buffer solution added with a salt with a molarity ranging from 0 to 0.5 M representing a linear gradient of pH 7.6 to 4.0.
• a satisfactory elution rate is of the order of 20 ml / h.
• the active fractions recovered are chromatographed on CM-Tnsacryl equilibrium in a buffer of pH 4.2, in particular a sodium acetate buffer 0.04 M.
• the active fractions are dialyzed against the acetate buffer beforehand, then deposited on the CM-Trisacryl column, rinsed with this same buffer.
• a first buffer of pH 5, 0.1 M is used, which can be carried out in 1 hour.
• a second buffer, of pH 4.2, 1 M makes it possible to elute the majority of the SCLs. This operation can be carried out in about twenty minutes.
• dialysis is advantageously carried out to remove the dialyzable molecules using a 0.01 M Na phosphate buffer, pH 7.2.
• Sugar constitutes the ligand of an agarose gel affinity chromatography column, in particular of hexamethylene-polyacrylamide-agarose diameter.
• the gel is first washed with a 0.5 M buffer, for example NaCl, then distilled water and finally centrifuged at low speed so as to get rid of the non-adsorbed substances.
• a sugar solution, pH 4, for example 0.1 M N-acetyl neurammic acid is added to the gel, followed by that of an agent such as a carbodimide.
• the pH is maintained at a value of the order of 4.5 and 5 at room temperature for approximately 1 hour, then is subjected to gentle stirring for 10 to 15 hours.
• the gel is washed several times in order to remove the retained impurities.
• the column filled with this preparation is balanced to pH 7.2.
• the use of 0.01 M sodium phosphate buffer is appropriate.
• the active sample from the previous step is deposited on the column and advantageously is equilibrated beforehand with the phosphate buffer.
• the first buffer (I) advantageously consists of a 0.01 M sodium phosphate solution pH 7.2 containing 0.15 M NaCl. It leads to the production of a peak which contains the majority of the SCL.
• the second buffer (II) also contains ethylene glycol, in particular at a rate of approximately 40 to 60%, preferably 50%. It leads to another peak which contains the majority of impurities.
• the column is then rinsed with the sodium phosphate buffer.
• the recovered SCL is analyzed by HPLC using a conventional water / acetonitrile / trifluoroacetic acid system and the fraction corresponding to the main peak is then analyzed by SDS-PAGE.
• the invention thus provides the means to isolate products of very high purity, and to have a product that is almost completely free of albumin.
• SCLs do not act directly on interferons, but inhibit the synthesis of secondary effector proteins induced by interferons.
• the number of these induced effector proteins is variable from one interferon isoform to another. Furthermore, the same molecular form does not necessarily induce the same effector proteins from one cell to another.
• SCLs restore to the cell its initial state. As a result, cells recover their ability to respond to growth stimuli, which would otherwise be inhibited by interferons. 2.
• Direct growth stimulation is obtained in the absence of serum and involves a large number of cells, immunocompetent or not. This stimulation is obtained without retro-inhibition effect which results in the development of the refractory state of the cells to repeated inductions of this substance.
• the SCLs of the invention can be administered repeatedly, if necessary in combination with specific growth factors, with which they can act in synergy.
• An example is interleukin 2 (Il 2), which cannot stimulate the proliferation of T lymphocytes, unless these lymphocytes are activated beforehand by a lectin or another antigen. Sarcolectin could then act as a physiological activator of the Il 2 receptors.
• sarcolectin will be associated with different interleukins or growth factors for a targeted amplification of growth, thanks to the resulting synergy. 3.
• the mechanism of carcinogenesis seems to be at least partially clarified.
• Proto-oncogenes are genes involved in the normal process of proliferation, either as a growth factor, either as a corresponding receptor, or by intervening in the metabolic chain involved in the growth process.
• the SCLs of the invention especially the SCLs corresponding to SEQ ID No. 3 or SEQ ID No. 4, or the derived sequences, are involved in the initiation of the synthesis of cellular DNA preparing the action of the factors of specific growth.
• the 55 kD SCL described above is a constitutive glycoprotein which is excreted in the extracellular medium, and stimulates growth in a non-specific manner.
• sarcolectins and growth factors As the effect of sarcolectins and growth factors is additive, we can consider it as a co-oncogene, on the one hand by its own action on cell proliferation and its synergy with growth factors, on the other hand, by its inhibitory effect on the antiproliferative functions of interferons. As with all lectins, the biological functions of sarcolectin are inhibited by specific sugars. In view of the properties reported above, the invention relates more specifically to the following applications of SCLs.
• the invention relates to their use as growth co-factors, to contribute to the growth of tissues, in particular to contribute to the regeneration of damaged tissues and to the acceleration of scarring.
• they constitute highly effective therapeutic agents, of local or general application.
• growth factors they can be used for cell cultures in vitro.
• Particularly preferred products in this regard are constituted by human recombinant SCLs. Indeed, when cultivating lymphocytes freshly taken from a healthy individual, one can stimulate their proliferation in the presence of interleukin 2. After successive passages in Hl medium without serum, the cells replicate in the presence of only 112. But the only protein excreted in the medium is SCL, in a dimeric form.
• Tests carried out by adding SCL in an appropriate amount show its favorable effect on cell growth.
• the invention therefore also relates to the use of SCLs as therapeutic agents for stimulating the immune system, in particular as a stimulant of specific immunity, where appropriate SCLs in association with an antigen, for example with a growth factor such as 1 'interleukin.
• SCLs can activate the synthesis of receptors for interleukins (They), in particular 112.
• the continuous expression of SCLs in the cell appears to inhibit the refractory state cells with repeated interferon inductions, leading to continuous production of interferon at each induction with however an inability of the cells to express IFN functions.
• SCLs or their inhibitors are thus advantageously used in protocols for repeated administration of IFN, for treating pathological conditions of infectious origin, for example during the final stages of AIDS infections or during auto-diseases.
• immune systems such as lupus erythematosus.
• the addition of recombinant SCL causes agglutination and the synthesis of type 1 or 2 interferon.
• the SCLs of the invention can also be used as vaccination adjuvants, because of their ability to increase the proliferation of immunocompetent cells.
• the invention relates to the use of the SCLs of the invention as tools for searching for compounds which inhibit natural sarcolectins.
• the invention relates to the use of SCLs to select compounds that inhibit by competition of their lectmic activity, antibodies or sugars, or even antagonists such as butyric amino acids or other butyroids.
• the SCLs are, where appropriate, fixed to albumin for stabilization purposes, to produce a retarded vehicle or to facilitate their diffusion in the tissues and the expression of their functions. .
• Inhibiting sugars are specific sugars present on the cell membrane. These are simple or compound sugars, such as N-acetyl galactosamme, sodium galacturonate, acetylated sugars such as N-acetyl neurammic acid, lactose alpha or beta, galactose, neuramme-lactose.
• simple or compound sugars such as N-acetyl galactosamme, sodium galacturonate, acetylated sugars such as N-acetyl neurammic acid, lactose alpha or beta, galactose, neuramme-lactose.
• Amy butyric acids include alpha-ammo butyric acid, alpha-ammo isobutyric acid and gamma-ammo butyric acid (GABA). These compounds have a high affinity in particular for transformed cells and inhibit the grafting of Sarcomas TG180 cells (or other cells) in mice.
• GABA gamma-ammo butyric acid
• the inhibitor compounds make it possible to considerably increase the antiviral resistance induced by IFN and are used with advantage in protocols comprising treatments with IFNs. They can also be used in anti-cancer therapies.
• the invention relates in particular to the use of these inhibitors in such treatments in combination with immunomodulators such as
• the anti-SCL antibodies of the invention are particularly valuable agents for inhibiting the effects of SCL produced in excess in pathological conditions such as cancers, chronic viral or autoimmune diseases.
• the drugs produced from these antibodies are characterized in that they contain an effective amount of these antibodies for the intended applications, in combination with an inert pharmaceutical vehicle.
• these drugs are especially suitable for anti-tumor treatments.
• these antibodies can be used for all immunological reactions, in particular ELISA and Western Blots, and make it possible to qualitatively and quantitatively determine the presence of SCL in a biological extract taken from a patient.
• the invention thus relates to a method for detecting m SCL in vitro, and especially the 55 kD SCL as purified according to the methods described. above, or corresponding to the protein expressed by SEQ ID No. 1, or also obtained by synthesis.
• This method includes: - bringing a biological sample to be analyzed from a patient or cells into contact with an anti-SCL antibody preparation or a Fab fragment immobilized on a solid support under conditions suitable for production of an antigen-antibody complex with the SCLs when they are present in the sample or the cells, then
• This detection method makes it possible to reveal, with great sensitivity and quickly, the presence of SCL in the test sample, the revelation of the possible reaction of antigen-antibody type.
• the invention also relates to a kit which can be used to carry out such detection.
• This kit is characterized in that it comprises: an appropriate solid phase serving as a support
• the detection relates to the presence of genes coding for the SCLs and comprises
• the invention also relates to a kit which can be used in this method and comprises said probes as well as the buffer solutions and reagents useful for carrying out the hybridization reaction.
• SCL inhibitors of the invention consist of the antisense nucleotide sequences defined above. These antisense blocks the expression of SCL, for example in the case of osteogenic sarcomas.
• SCLs according to the invention are based on their capacity to agglutinate cells and their affinity for simple sugars. These properties are used in diagnosis or in therapy. Other characteristics and advantages of the invention will be given in the examples which follow.
• FIGS. 1 to 5 represent respectively - FIG. 1, the elution profile of the SCL fractions chromatographed on DEAE-cellulose, obtained after chromatography on Sephacryl of tissue extracts treated beforehand,
• FIGS. 2A and 2B the elution profiles of the fractions obtained by chromatography with a sugar ligand and the analysis in reverse HPLC of the active fraction
• EXAMPLE 1 SARCOLECTIN PURIFICATION PROCEDURE The diagram of this procedure is as follows: Stage I - Preparation of biological material and freeze-drying.
• Stage II Hydration of the material and pretreatment either with pepsin or at pH 5.
• Stage III Chromatography on Sephacryl S-200.
• Step V Affinity chromatography on sugar.
• Step VI- Chromatography in reverse phase HPLC (for sequencing): column C18 gradient H 2 0 / acetonitrile column C4 gradient H 2 0 / acetonitrile
• the supernatant which contains the sarcolectin is collected after filtration on sterile gauze and on a 1.2 ⁇ millipore filter, distributed in 2.5 ml pill boxes, then lyophilized before storage at -80 ° C.
• the preparation is then subjected either to a treatment with pepsin, or to a treatment at pH 5 (isoelectric point of SCL).
• the tissue extracts (concentration 6%) are treated with a solution of pepsin crystallized twice (2,675 units / mg) at the final concentration of 1 mg / ml.
• the reactive mixture is adjusted to pH 2 (optimal pH of the activity pepsic) and incubated at 37 ° C for 2 h; a large precipitate is formed.
• the enzymatic action is then stopped by adjusting the pH to 7.3 and addition of Iniprol (Choay) IO 5 units / mg protease overnight. at 4 ° C.
• the sample is centrifuged and the supernatant removed is then filtered on Sephacryl S-200 gel.
• a column (Pharmacia 2 x 30 cm) which contains the Sephacryl S-200 gel (Pharmacia) previously swollen in buffer.
• the sample is introduced into the column in a volume of 2.5 ml, the flow rate of the eluent (PBS) is 20 ml per hour and the volume of each fraction is 1.35 ml.
• the determination of the molecular weight of the filtered sample is calculated by reference to a range of proteins of known molecular weights.
• the molecular weight estimate is based on the linear relationship between the effluent volume (Ve) and the logarithm of the molecular weight.
• a column is filled with diethylammoethylcellulose (DEAE 52, Whatman, England) previously swollen and balanced in the following pH 7.6 buffer: NaCl
• the sample (pool of biologically active fractions obtained after filtration on Sephacryl S-200) is introduced into the column which is then carefully rinsed with the same buffer.
• the proteins are eluted for 4 h in the presence of buffer supplemented with NaCl of varying molarity from 0 to 0.5 M representing a linear gradient from pH 7.6 to 4.0.
• the elution speed is 20 ml / hour.
• the recorded profile is shown in Figure 1. Peak III contains albumin and traces of SCL, while peak IV (hatched) corresponds to biological activity and contains the majority of SCL. The latter is eluted with a 0.25 M sodium buffer.
• This step can be replaced by chromatography on a Mono Q column in HPLC (L-histidme gradient 20 mM pH 5.5, 6.0--0.1 NaCl).
• a Pharmacia column (1 x 15 cm) is filled with CM-T ⁇ sacryl-M (IBF, France) then equilibrated with 0.04 M sodium acetate buffer pH 4.2.
• the sample containing the active fractions of sarcolectin is previously dialyzed against the 0.04 M acetate buffer pH 4.2 and then deposited on the column which is then rinsed with the same acetate buffer for 1 h.
• the elution profile includes two buffers:
• the first buffer 0.1 M sodium acetate, pH 5 which mainly eliminates albumin; the elution lasts about an hour; (see FIG. 1 A where peak 15 contains albumin and peak 50 (hatched peak) the SCL expressed in cytoagglutmating activity (CA).
• CA cytoagglutmating activity
• Fractions 40-50 contain sarcolectin and are dialyzed against the 0.01 M Na phosphate buffer pH 7.2 which will be used for the next step.
• the conventional water / acetonitrile / trifluoroacetic acid 0.1 l system is used, with detection at 220-280 nm.
• the gradient is programmed as follows:
• the corresponding SDS-PAGE fraction is analyzed in SDS-PAGE gel.
• the Western blot shows the three bands using the ant ⁇ -55kD serum: a 65 kD, 55 kD and ⁇ 14 kD.
• the use of ant ⁇ -65kD serum is hampered by the fact that it is constantly contaminated with albumin.
• the different stages of the purification are followed with respect to two characteristic functions of the SCLs, namely their capacity 1) to stimulate the synthesis of DNA in human T H9 cells, cultivated in a medium devoid of serum for 24 h and 2) to agglutinate cells.
• the results for the last 3 purification stages are reported in table 1, indicating the count per minute (CPM) of thymidine 3 [H], the percentage of stimulation and the cellular agglutination (unit / 0.05 ml).
• the purification is of the order of 16,500 times.
• the purified protein obtained at the end of the procedure described above gives a unique peak after analysis by HPLC.
• the ant ⁇ -65 kD serum Due to the proximity of their size and their close physicochemical properties, the ant ⁇ -65 kD serum also reacts with albumin. On the other hand, the ant ⁇ -55 kD serum appears to be completely homogeneous. Both the anti-65 kD serum and the anti-55 kD serum recognize the proteins of the other two molecular weights, after analysis by Western blots (FIG. 3B, where tracks 1 and 3 correspond to the use of anti-65 kD antibodies and lanes 2 and 4, to that of anti-55 kD antibodies).
• Rapid purification can be obtained for certain biological extracts, for diagnostic purposes
• the supernatant contains the proteins 65 and 55 kD recognizable by Western blots using specific antibodies.
• the titer can be estimated by ELISA using the same antibodies.
• FIG. 4B the Western blot using the same preparation intensely marks the minor band of 65 kD.
• a commercial cDNA library from 34 week human placenta is used. These cDNAs were cloned into the EcoRI site of the galactosidase gene from phage lambda gt 11. The selection was made using in parallel anti-sarcolectm antibodies obtained separately against the 65 kD protein and the 55 k protein: as described in Example 1. In summary, the strips were isolated and excised, lyophilized, and crushed to then be used to immunize rabbits. The anti-65 kD serum recognizes the corresponding protein and also a little albumin. On the other hand, the anti-55 kD antibody appears specific. The two antibodies were used in parallel, in a well-defined order. Table 2 gives a summary of the methods followed to isolate 4 clones.
• the library was amplified to obtain about IO 5-10 b clones.
• Empty clones produce galactosidase -1. They are colored blue after IPTG + X-gal treatment.
• the cloned genes are inserted into the EcoRI site of the galactosidase and the fusion protein thus obtained is colorless.
• the colorless colonies are then treated with specific sera to which an anti-lapm antiserum paired with the peroxidase revealed by DAB is added, which allows their revelation.
• the isolated cDNA is 1.8 kb long. It contains an open reading phase of 1,407 bp which contains the genetic information for 469 amino acids. Its structure is given on SEQ ID N ° l. The initiation ATG and termination TGA sequences are in position 62 and 1469 respectively.
• the A domain corresponding to approximately 320 bp in DNA is variable; it is translated into a ⁇ sheet in the protein.
• This region contains 12 blocks of 80 to 100% homologous to those found in the 14 kD lectme of the galactoside-binding protein (140/320 bp).
• This domain contains the essential part for the lectmic function of the molecule. In total, at least 30 of these blocks are located mainly at the ends.
• the domain B is formed by 4 ⁇ helices. It has the following homologies for DNA:
• Human keratin 56 Kd 78% homologous over a length of 814 bp.
• Human vimentme 52% homologous over a length of 625 bp
• Human neurofilament homologous to 553 over a length of 589 bp.
• Domain C contains short sequences translated into a ⁇ sheet which also has lectin sequences.
• C. Glass et al. (9) have isolated a gene and have classified this molecule as an intermediate filament under the name of mesothelial cytokeratme. This identification is based on the sequence analogies cited above and which relate to the ⁇ -helix sequences stable to the molecule.
• variable 5 ′ segment forming the ⁇ sheet contains the lectmic function of the molecule.
• the stable domain of the four ⁇ helices which occupy the B domain seems to ensure the stability of the molecule.
• the biological significance of this 55 kD molecule is fundamentally different from that taught by the previous authors for the molecule they have described.
• the SCL of the invention is excreted in the medium. This property is unusual for the intermediate filaments, which are part of the cytoskeleton, have the essential role of contributing to the stability of the intracellular structure. SCLs are expressed in monomeric form, or in some cases dimé ⁇ que.
• the intermediate filaments are, on the contrary, polymerized into multimeres.
• a wide variety of cells from either rodents or primates have been tested. Agglutination is obtained by suspending the cells in MEM medium in the absence of serum.
• the test can be carried out in two ways, either in the presence of a dilution range of sarcolectin in geometric progression based on 2, by seeking the concentration at which 50% of the cells are agglutinated, or by seeking the affinity of the different sugars. for cellular receptors.
• the munne sarcolectm from liquid ascites of Swiss mice grafted with Crocker's TGI80 sarcoma contains 32 units agglutinating the H9 cells which originate from T lymphoma, fixed by 10% formalin.
• the table below illustrates the affinity of the receptors for the various sugars in the presence of 2 agglutinative units.
• the affinity is higher for ⁇ lactose than for a lactose, while the opposite is observed with SCLs originating from human placenta.
• the highest affinity for human tissue is N.A.N.A.
• Other inhibiting sugars include N-acetyl-glucosamine or even galacturonate (especially for hamsters), it being understood that this list is not exhaustive. The inhibiting sugars not only prevent the agglutination of cells, but also hinder the various biological effects of sarcolectins, in particular the stimulation of growth.
• mice Male mice are injected (average weight:
• the invention makes it possible to select those of sugars exhibiting the desired inhibitory effect and to develop medicaments containing them as active ingredients in the appropriate quantities with a view to highly effective antiviral therapy.
• SCLs can act in synergy with growth factors and promote cell multiplication indirectly. Natural SCLs could therefore promote oncogenesis by blocking the effect of interferon. We will therefore measure the advantage of inhibiting the action of natural SCLs to promote the balance of tissue development in favor of growth inhibitors.
• TG-180 tumor cells were grafted, originating from a tumor resistant to chemical metabolic inhibitors. At the concentration of 3 x 10 b cells injected by the IP route into Swiss mice of 20 gr, an ascites tumor is obtained which is detectable in 10 days and kills practically 100% of the animals in 21-25 days.
• mice 200 ⁇ g / mice, or interferon, or both.
• mice 4 groups of mice were used per experiment. The results obtained are given in table 4. They are estimated by the percentage of tumors which appeared on the 10th day, by the mean time of animal survival (MST) in days, and the final survival (in percentage). TABLE 4
• gamma amino-butyric acid and alpha amino-isobutyric acid significantly inhibit tumorigenesis.
• the onset of tumors is delayed, the increase in average survival is obtained in all groups.
• a single injection of CP also allows a significant increase in the final survival of the animals, which is further amplified with the association of interferon.
• the SCLs of the invention constitute particularly valuable models for the study of the anti-tumor effects of amino butyric acids and the development of a treatment protocol (Table 5).
• H9 T lymphocytes and Daudi B lymphocytes H9 T lymphocytes and Daudi B lymphocytes, U937 monocytes, and studied in parallel, normal T and B lymphocytes of splenic origin, murine L929 cells and human HeLa cells.
• the growth medium did not contain serum, only highly purified sarcolectin as obtained according to Example 1.
• the antagonistic effect of sarcolectin on the action of mterferon can be estimated by treating the cells with interferon for 5 to 6 hours, then removing the medium and replacing it with sarcolectin for 18 hours.
• the antagonistic effect of SCL appears from the 5th hour and can lead to the restoration of the cell to the initial state before treatment with interferon.
• Sarcolectin can also 3D inhibit the action of IFNs induced either by poly (I) (C) or by Newcastle virus.
• Diagnosis requires a rapid method of purification.
• the level of sarcolectin in human or animal serum can be obtained for example as follows: dilution of the serum to 1/10;
• the purified preparation contains only a 65 kD band. However, at high concentrations, a 55 kD band can be detected, using Western blot using monoclonal or mono-specific anti-sarcolectin antibodies.
• H9 cells originating from human T lymphomas treated with formalin at 10 °.
• the cells can be maintained in a phosphate buffer without special precautions.
• the titer of sarcolectin can be estimated by a range of geometric dilutions, generally based on 2, in a microplate to which a suspension of fixed cells is added. Agglutination occurs at 4 ° C and the limit is estimated by the dilution at which approximately 50% of the cells are agglutinated.
• the suspension of cells to be tested must be incubated with the quantity of tritiated thymidme arbitrarily chosen. It is important that the medium does not contain serum during the test period, which is generally 24 hours.
• This application is based on the analysis of the development of tissues whose growth is normal and rapid like the fetus.
• the placental blood contains on the one hand, growth factors and SCL, which stimulate DNA synthesis, and, on the other hand, interferons which, on the contrary, inhibit it by promoting cell differentiation. These three types of factors appear alternately, which results in discontinuous growth.
• Recombinant interleukin 2 can also be combined under comparable conditions.
• the target treatment includes IFNs, especially of the ⁇ or ⁇ group injected every 48 h by the parental route (at doses in general of 3-5 x IO 6 units per injection ) for a month.
• This treatment provides cells generated in phase 1 with better differentiation.
• the effect of IFNs can be considerably increased by the butyric amino acids (5g / kg) tested above.
• interferons can be amplified by the combination of the sugars mentioned above, in particular lactoses ( ⁇ or ⁇ ), D galactose, N-acetyl neurammic acid (NANA) chosen as example.
• lactoses ⁇ or ⁇
• D galactose D galactose
• NANA N-acetyl neurammic acid
• Their nature can vary depending on the species or tissue. For example, in osteogenic sarcomas in children, local treatment based on these notions could usefully complement surgical treatment, by combining SCL -aspartate, followed by IFN, ⁇ lactose, NANA
• the cloned molecule was introduced into a plasmid containing 2 promoters, one of them being a transcription promoter inducible by hexamethasone.
• the choice of this system is justified by the fact that SCL is practically present in all the cells studied so far and could be at the origin of the functions present in the host cell.
• Mouse L cells were transfected with the plasmid containing the cloned gene.
• the anti-interferon function has been studied by the ability of interferon to inhibit the multiplication of a developer: the vesicular stomatitis virus.
• the interferon function was then quantified in mouse cells (use of variable concentrations of interferon and search for the limit dilution at which the antiviral state is completely inhibited).
• the vesicular stomatitis virus destroys the entire cell population in virgin cells.
• the sensitive cells were treated with 200 IU of interferon for 5 hours. Decreasing amounts of SCL, diluted on a geometric scale based on 2, were then added. In the example chosen, the 1/32 dilution was the limit at which the action of interferon was completely blocked, viral multiplication is normally resumed.
• the antibodies induced respectively by the peptides SEQ ID No 5 (oligopeptide 41-55 in SEQ ID No 1) and SEQ ID No 6 (oligopeptide 81-95 in SEQ ID No 1) are specific for each of the peptides.
• the SCLs excreted by the osteogenic sarcoma ESS are strongly recognized by the anti-peptide antibodies 41-55.
• the SCL is excreted in H-1 medium by PBMC in 24 h maintained without serum.
• the results obtained are given in Table 8.
• SCL is the only protein detected by electrophoresis after Coomassie blue staining and identified by known Western Blot monoclonal antibodies being SCL.
• CAGCCACCAC CCACAATCAC AGCCATTGCC GAGGCTGAGG AGTGTGGGGA GCTGGCGCTC 1556
• GAGATCGCCA CCTACCGCAA GCTGCTGGAG GGCGAGGAGA GCCGGTTGGC TGGAGATGGA 1736

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