EP0865491A1 - Compounds having lectin properties and biological uses thereof - Google Patents

Abstract

Nucleotide sequences capable of coding for polypeptides having lectin properties and the corresponding sarcolectin-type polypeptides, and therapeutical uses thereof, are disclosed. In particular, the use of sarcolectin-type polypeptides for stimulating immunity, optionally in combination with interferon or butyroids, is disclosed. The use of specific inhibitors or antagonist peptides enables sarcolectin production to be opposed. Antibodies to said peptides are suitable for diagnostic and therapeutical use.

Description

 Compounds with lectinic properties, and their biological applications.

The invention relates to compounds having lectin properties, and to their biological applications.

It relates more particularly to proteins or polypeptides of the sarcolectin type.

We know that this term was used to designate the lectmes identified in human osteogenic sarcomas or those induced in the hamster.

(after inoculation of Moloney's sarcoma virus), where they are present in high levels. But sarcolectins are also found in a wide variety of normal or tumor tissues in vertebrates (primates, rodents, gallinaceous).

They can also be detected on the surface of normal or transformed cells of human or animal origin.

Purified sarcolectin preparations have already been described and their properties reported.

The most regularly characterized physicochemical properties are: sensitivity to proteases (trypsin,

Pronase ^R ), but also resistance to mild treatment, under certain conditions, with pepsin; resistance to thermal variations

(-20 ° C to + 100 ° C);

- resistance to variations in pH (2 to 8);

- resistance to detergents such as SDS and dithiothreitol;

- The migration in SDS-PAGE gel of the protein in the PM region of 65-55 kD. Their biological properties are of three types (see references (1) to (8) given at the end of the description, with the other references cited below, in the document "Bibliographical references"): - agglutination of normal or transformed cells ( activity at the level of the cell membrane). Cyto-agglutmation can be inhibited by the affinity of sarcolectins for simple sugars. - stimulation of the growth of human T and B lymphocytes, Daudi lymphoid cells, and cells adherent to the substrate, such as L929 mun cells, transformed rat fibroblasts (Fr3T3) and human fibroblasts (FS4). Thus, sarcolectins behave as promoters of cell growth, of specificity not determined;

- reduction or abolition of the antiviral state pre-established by interferon (IFN) and restoration in the cell of the initial sensitivity to the virus. After establishing antiviral resistance, 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. When the cell is restored, it is possible either to stimulate growth by the SCL or by other growth factors, or on the contrary to reprocess the cells by the IFN to develop again the antiviral resistance. 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.

It will be emphasized that all the attempts prior to the invention have retained the 65kD protein as a molecule comprising the biological properties of SCL. During the identifications based on the acrylamide gel electrophoreses followed by Western Blot, 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. Surprisingly, it appears that 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.

The term 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.

According to another aspect, 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.

The 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".

By 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). .

Such 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. These 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. 1. In this sequence, 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. In this regard, 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.

The various 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.

The corresponding mRNA sequences, or even the antisense sequences corresponding to the sequences defined above, as well as the sequences complementary to these different sequences, also fall within the scope of the invention. These different nucleotide sequences are further characterized in that they are free of mammalian DNA, infectious agents, prions and other materials from natural products. As indicated above, the 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.

These modifications make it possible, if desired, to adapt the sequences defined above to expression in a type of vector or host, or even to facilitate cell penetration of the encoded polypeptide or to increase its activity.

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.

These proteins do not contain any element likely to be confused with albumin.

The invention relates in particular to SCLs having a chain of amino acids in SEQ ID No. 3 or in

SEQ ID N ° 4.

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. 3, in particular those of the 5 ′ region corresponding to SEQ ID No. 4, are essentially responsible for the lectmic function of the SCLs defined above.

Other particularly preferred 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.

These are peptides having the sequence SEQ ID No. 5 or peptides derived from this sequence, and 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.

These are peptides having the sequence SEQ ID No. 6 or peptides derived from this sequence, and characterized in that the antibodies directed against this fragment do not recognize the fragment 41-55 above.

It will be recalled that by derived peptides is meant 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.

Other SCLs of the invention are fragments or derivatives obtained by modification of the sequences SEQ

ID N ° 3 or SEQ ID N ° 4, as long as they retain at least part of the lectin properties defined above.

As indicated in relation to the nucleotide sequences, 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:

- treatment of the 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,

- chromatography on Sephacryl-S-200,

- chromatography on an ion exchange resme such as DEAE-cellulose or CM-Tπsacryl, - affinity chromatography using as sugar ligand, such as neurammic N-acetyl acid, the purity of the SCLs being verified by reverse HPLC.

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.

It has structural and functional properties that distinguish it from the classic group of intermediate filaments, although it is recognized by a monoclonal anti-cytokeratin mesothelial type 7 antibody.

Indeed, it is in monomeric form, if necessary dimeric, while in hindrance the intermediate filaments are polymerized into tetramers or more. It is an extracellular protein, excreted m vivo as in vitro while the intermediate filaments are intracellular or participate in the maintenance of the structure.

It has lectmic properties namely an inhibitory function of the actions of IF ^" has the capacity to stimulate cell synthesis and to agglutinate cells. These properties are essentially conferred by the 5 'end, as evidenced by digestion by pepsin which can abolish the stimulative function of DNA synthesis, with conservation of size and sedimentation in SDS-PAGE.

By providing a highly purified SCL, 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.

To obtain the nucleotide sequences, it is possible to operate, according to conventional techniques, synthetically. As a variant, in particular in order to obtain at least part of the sequences of the type SEQ ID No 1 or SEQ ID No 2, 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.

To obtain the SCLs of the invention, 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.

By applying the conditions required to obtain the expression of the SCL sought for lectmic activity, 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.

For the production of SCLs, 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,

- recovery of the fraction containing most of the lectmic activity, as measured by the cell agglutination test, - the passage of this fraction on ion exchange resins, in particular DEAE-cellulose and CM-Trisacryl, the passage of the fraction containing the essential of the lectmic activity, on a column containing as ligand a sugar such as N-acetyl neuraminic acid.

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.

The extract thus treated is subjected to a succession of chromatography steps. 1 - Chromatography on Sephacryl

Preferably, 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.

2- Chromatography on DEAE-cellulose The sampled fractions are collected and the fractions with the maximum lectmic activity are subjected to chromatography on DEAE-cellulose before swelling and equilibrating with a buffer solution of pH close to neutrality, in particular pH of the order of 7.6.

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.

3- Chromatography on CM - Tπsacryl

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.

Preferably, the active fractions are dialyzed against the acetate buffer beforehand, then deposited on the CM-Trisacryl column, rinsed with this same buffer. To remove albumin, a first buffer of pH 5, 0.1 M is used, which can be carried out in 1 hour. The use of 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. Before the active fractions are brought into contact with a sugar, dialysis is advantageously carried out to remove the dialyzable molecules using a 0.01 M Na phosphate buffer, pH 7.2.

4- Affinity chromatography using a sugar ligand: 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. Advantageously, 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.

For example, 1 M NaCl is used, then 0.1 M acetic acid, and finally bi-distilled water.

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.

After rinsing the column with this buffer, the elution is carried out with at least two buffers, one to elute the SCL, the other to remove the other proteins and regenerate the column. 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.

5 - Purity control

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.

The study of the purified SCLs thus obtained and of the SCLs corresponding to the expression products of the nucleotide sequences defined above shows that they have lectinic properties of great interest.

1. These 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. By inhibiting the synthesis of all IFN-dependent secondary proteins, 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.

Therefore, unlike the usual immunostimulants, 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. In other examples, 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. It is generally accepted that malignant transformation results from a process which, in successive stages, starting from benign proliferation, would lead to the selection of highly carcinogenic cells. 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. In particular, 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. 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.

Thus, 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.

In this regard, they constitute highly effective therapeutic agents, of local or general application. As 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 the only protein excreted in the medium is SCL, in a dimeric form.

In the H9 continuous T lines, cell multiplication is ensured by the SCL produced by the cells and excreted. In fact, in both cases, it is the only major protein detected in the medium by SDS-PAGE and Western blot.

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.

Thus, as physiological activators of the proliferation of T lymphocytes, 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.

The invention relates in particular to their use in interferon treatments by taking advantage of their inhibitory effect on the synthesis of secondary effector proteins so as to restore the cell to its initial state and the normal sensitivity to

1 interferon.

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. In certain competent cells, in particular those of PBL, 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.

According to another aspect, the invention relates to the use of the SCLs of the invention as tools for searching for compounds which inhibit natural sarcolectins.

In particular, 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. In the various therapeutic applications mentioned above, 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. .

Medicines produced from the inhibitor compounds mentioned above, which contain effective amounts of these compounds to obtain the desired inhibition, in combination with pharmaceutical excipients, also fall within the scope of the invention.

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.

These sugars, by binding to the cell membrane, interfere with the binding of SCL. On the basis of these observations, complex butyric derivatives can be obtained by fixing galactose, lactose, NANA, glucosamme, galactosamme, N-acetyl galacturonic acid on these butyric derivatives or on other butyric derivatives such as esters containing, for example, octal butyrate, or PEG butyrate. (PEG = polyhylene glycol).

Overall, the majority of these inhibitors indirectly increase the expression of induced interferons, increase the expression of endogenous interferon during the different phases of normal growth or the process of tumogenesis. Butyroids can be used as growth inhibitors; they give a mterferon-like effect, but by different mechanisms. Their effect is not directly inhibited by SCLs, but acts as an antagonistic effect of SCLs by different mechanisms.

The butyric derivatives formed of ¹ hydrophobic-hydrophilic amino acids such as 1-valme t-butyl ester, by their hydrophilic functions, bind to the cells and by their hydrophobic functions, maintain the molecules in contact with the membrane.

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.

Thanks to these inhibitors or antagonists, excessive or continuous antiphysiological productions of interferons can be re-equilibrated (by inhibiting the action of SCL or by affixing their effects using antagonists) to fight against immune disorders induced by certain chronic viral infections (HIV), or immune diseases.

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

Corynebacterium parvum, which SCL can replace if there is no constitutive production of SCL.

Other products capable of inhibiting by competition the activity of the SCLs of the invention are constituted by antibodies. These polyclonal or monoclonal antibodies directed against the SCLs of the invention are advantageously produced according to conventional methods. They are also targeted by the invention as new products.

In therapy, 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. In diagnosis, 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

- Demonstration of the formation of such an antigen-antibody type complex, operating advantageously according to the usual techniques. Cytofluorometry techniques are used, for example.

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

- a preparation of anti-SCL antibodies or Fab fragments, free or immobilized,

- buffer solutions and reagents suitable for immunological reactions and for detection reactions. The antibodies used in these methods and kits are advantageously antibodies directed against the peptides SEQ ID No. 5 defined above or their derivatives. As a variant, the detection relates to the presence of genes coding for the SCLs and comprises

- carrying out the step of bringing the biological sample to be analyzed or of cells originating from a patient into contact with a probe as defined above, under conditions suitable for the production of a hybridization complex, when the genes coding for the SCL are present in the sample or the cells,

- the demonstration of the hybridization complex and the quantification of the expression of SCL by these genes.

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.

Still other 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.

Still other applications of 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.

Reference is made thereto to FIGS. 1 to 5 which 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,

- Figures IA and IB, the elution profiles on CM-Trisacryl of the biologically active fractions of

SCL collected on DEAE-cellulose,

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,

- Figures 3A and B, control by an SDS-PAGE gel of the different stages of purification and the Western blot using anti-65kD and anti-55kD antibodies. - Figures 4A and 4B, a photo of

SDS-PAGE electrophoresis of an SCL preparation before and after gentle pepsin treatment and of a Western blot showing the reaction of anti-65kD monoclonal antibodies with the protein of the invention and albumin respectively, and FIG. 5, the photo of a Western blot using the anti-55kD antibody to study different samples.

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.

Stage IV - Chromatography on ion exchange resin: DEAE-cellulose or CM-Trisacryl

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

I) Preparation of the biological material and lyophilization The tissues removed are washed carefully in minimal Eagle medium until the elimination of all traces of blood, then are lyophilized and stored in the dry state at -20 ° C. The dry tissue is then cut finely until a powder is obtained which is weighed and hydrated in MEM medium (supplemented with antibiotics) at a concentration of 6% (dry weight in g). The suspension is stirred at + 4 ° C. for 20 h, centrifuged at low speed 4000 rpm for 20 min for clarification, then centrifuged at 27,000 rpm for 2 h at 90,000 g (advantageously a Spinco L3 ultracentrifuge is used).

The supernatant which contains the sarcolectin is collected after filtration on sterile gauze and on a 1.2 μ millipore filter, distributed in 2.5 ml pill boxes, then lyophilized before storage at -80 ° C.

II) Hydration of the material and pretreatment At the time of use, the lyophilized product is hydrated in 2.5 ml of double-distilled water, then well dissolved.

The preparation is then subjected either to a treatment with pepsin, or to a treatment at pH 5 (isoelectric point of SCL).

A) Pepsin treatment

It has been noted that a household treatment with pepsin makes it possible to preserve the physicochemical characteristics (migrations in the gel), and does not destroy the biological activity of the tissue extracts, but destroys other proteins contaminating the preparations.

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.

B) Treatment at pH 5 The preparation of tissue extract is adjusted to pH 5 by addition of IN HCl, then left at room temperature for 15 mm. A large precipitate appears which is centrifuged at 4,000 rpm for 15 mm. The supernatant once decanted represents the sample for the filtration step on Sephacryl S-200 gel. III) Chromatography on Sephacryl S-200

A column (Pharmacia 2 x 30 cm) is used 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.

The biological activity (cell agglutination capacity) of each fraction is then tested. The effluent volume of the fraction, which has the maximum biological activity, makes it possible, after plotting on the calibration curve, to determine the molecular weight which is between 190 and 200 kDa. IV) Chromatography on exchange resin

. DEAE cellulose

A column is filled with diethylammoethylcellulose (DEAE 52, Whatman, England) previously swollen and balanced in the following pH 7.6 buffer: NaCl

20 mM, EDTA 0.1 mM, β-mercapto-ethanol 15 mM, tris-acetic acid 20 mM. 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).

. CM-Trisacryl

A Pharmacia column (1 x 15 cm) is filled with CM-Tπsacryl-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).

- the second buffer: 1 M sodium acetate pH 4.2 which elutes mostly sarcolectin; elution lasts 20 minutes (4 mm./fraction) (see figure IB)

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. V) Affinity chromatography, using as ligand N-acetyl-neurammic acid (NANA for short).

9 ml of Ultragel-HMD-Aca 34, IBF code 2461 61 (hexamethylene-diameter polyacrylamide-agarose) are washed

3 to 4 times in 25 ml of 0.5 M NaCl buffer, then at least 2 times with bi-distilled water and finally centrifuged at low speed.

4 ml of a 0.08 M solution of N-acetyl-neurammic acid (type IV, Sigma) adjusted to pH 4.7 are added to the gel.

5 ml EDCL 0.1 M pH 6 (1-ethyl-3-3-dιmethyl-ammo-propyl-carbodιamιαe, Sigma) is then added and the pH is maintained between 4.5 and 5 for 1 h at laboratory temperature. It is then left under gentle stirring overnight. The gel is washed several times with 1 M NaCl, then with 2 volumes of 0.1 M acetic acid, and rinsed with bi-distilled water. A Pharmacia column (1 x 15 cm) is filled with this preparation and equilibrated with the 0.01 M Na phosphate buffer pH 7.2. The previously balanced sarcolectm sample is introduced against the Na phosphate buffer, and it is recycled three times. After rinsing the column with the same buffer for 1 h, the impurities not retained are removed and then the elution is carried out with two buffers:

Buffer E] _ = sodium phosphate 0.01 M pH 7.2 + 0.15 M NaCl which elutes sarcolectin (peak hatching in FIG. 2A) as indicated by the recording in DO and the titration of the biological activity (agglutination of cells by operating as described in - buffer E2 = 0.01 M sodium phosphate pH 7.2 + 0.15 M NaCl + 50% ethylene glycol to remove the other proteins (peak between 15 and 20) and regenerate the column. The column is then rinsed with 0.01 M sodium phosphate buffer pH 7.2. (Fig. 2A)

VI) Chromatography in reverse phase HPLC The apparatus used is an "HPLC Controller 2152" of brand LKB and the separation in reverse phase is carried out with a column C18 (Waters).

The conventional water / acetonitrile / trifluoroacetic acid 0.1 l system is used, with detection at 220-280 nm.

The gradient is programmed as follows:

Debit time% acetoni tril e

0 1 ml 0

40 1 ml 50

60 1 ml 80

70 1 ml 80

80 1 ml 0

A major peak located at 75% of the acetonitrile gradient is recorded (Figure 2B)

The corresponding SDS-PAGE fraction is analyzed in SDS-PAGE gel. The Western blot shows the three bands using the antι-55kD serum: a 65 kD, 55 kD and ≤ 14 kD. The use of antι-65kD serum is hampered by the fact that it is constantly contaminated with albumin. RESULTS OBTAINED

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).

TABLE 1

Examination of these results shows that the last three stages of the purification result in obtaining a practically pure protein.

Relative to the initial protein level, 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.

As for the ability to agglutinate these same cells, we see that it remains unchanged. After chromatography in SDS-PAGE gel stained with silver nitrate and denaturation, three characteristic bands are observed, the size of which has been successively estimated at 65 kD, 55 kD and <14 kD. (See lane 7 in FIG. 3A, where the other lanes correspond to the different stages of purification controlled by SDS-PAGE, namely the chromatographies 1) on Sephacryl S200, 2) on DEAE-cellulose, 3) on CM-Tπsacryl ( albumin peak 1), 4) corresponds to a size mark, 5) to CM-Trisacryl chromatography, (SCL peak 2), 6) to NANA affinity chromatography and 7) to HPLC).

These strips were cut, freeze-dried, ground and injected into rabbits for immunization.

Due to the proximity of their size and their close physicochemical properties, the antι-65 kD serum also reacts with albumin. On the other hand, the antι-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).

EXAMPLE 2: RAPID PURIFICATION METHOD

Rapid purification can be obtained for certain biological extracts, for diagnostic purposes

(for example, serum diluted to the tenth) by proceeding as

- Lowering the pH of the medium to pH 5 for 30 minutes; - A significant precipitate is then noted, which will be eliminated by centrifugation;

Readjustment to pH 7.4; 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.

In vivo, SCLs are found to be strongly linked to albumin, a concept which has been ignored until now. Using monoclonal antibodies, clearly detectable bands of this protein can be found by Western blotting (see Figure 4A:

SDS-PAGE electrophoresis colored by Comassie blue

- Below the major band of 67 kD containing albumin, there is a minor band of 65 kD. The gentle treatment of the preparation with pepsin digests the albumin, but retains the minor band, FIG. 4B: the Western blot using the same preparation intensely marks the minor band of 65 kD. We note the diffusion of the marker on

Albumin which is only indirectly labeled with the 55 kD protein.

These results were fundamental for going to

Against the interpretation which prevailed over the identity of the 65kD band. The invention thus made it possible to demonstrate that this molecule is in fact an artefact linked to the binding of a few 55kD SCL molecules on albumin. In Figures 4a and 4b attached to this text, this finding is clearly illustrated. It is also supported by the article by FU YUE ZENG et al. (12) in which the 5 'sequence of the SCL protein is considered to contain strong analogies with albumin. Similarly, using culture media which allow the growth of cells without serum, a unique protein band identified as SCL is detected, excreted from the cells: simian Vero, human osteogenic sarcomas, PBL T lymphocytes or cells. H9 in continuous culture. In these protein-poor cultures, SCL must play an important role in regulating growth.

EXAMPLE 3: CLONING AND IDENTIFICATION OF THE GENE OF HUMAN SARCOLECTINS

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.

. Identification of 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.

As indicated in Table 2, 10 clones were isolated during the first passage using the specific antibody (a). During the second passage, half of the descendants of each isolated clone were subcloned by the antibody antι-65kd (a) and the other by the antibody antι-55 kd (b)

This procedure made it possible to isolate, during the third cloning, a clone called 5, using antibody b, a clone named 6 with antibody b, a clone 9 with antibody b and finally, a clone 10 with l 'antibody a. This procedure therefore led to the isolation of a total of 4 clones. In the fourth cloning, only the anti-55kd antibodies (b) were used, and all the clones obtained, whether they were isolated by the antibodies (a) or

(b) have been recognized by the antibody b which is much more specific as indicated previously. Analysis of the sequences shows that in reality 3 different clones are isolated, designated clones 5 and clone β, because after sequencing clones 9 and 10, proved to be absolutely identical.

It will thus be observed that these three clones were isolated with the two different antibodies (a _/ and (b): their identity suggests antigenic relationships between the two sera, corresponding to epitopes shared by proteins. These proteins can therefore represent members from a family.

. Results obtained with clone 5 Structure and characterization of the clone

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.

EXAMPLE 4: STUDY OF THE 55kD PROTEIN

1) Two kinds of cell lines originating from reference human osteogenic sarcomas (MG 63) or HOS were cultivated. The culture of these cells was carried out in the presence of 1% fetal calf serum and RPMI medium. The proteins excreted in the medium after purification according to the method of Example 1 were analyzed. After Western blotting, a 65 kD band and a 55 kD band were obtained. Both proteins were excreted in the culture medium.

2) In a second series of experiments, these same cells were cultivated in a synthetic Whittaker medium (Ultra Doma MDCK) without serum, for two successive passages, so as to eliminate any residual albumin. In total, these environments do not contain very little protein (2-5 ng / ml). The cells excrete reads capable of agglutinating the H9 cells (T lymphocytes in continuous line treated with 10 ° of formalin). After Western blotting (using both monospecific antibodies against the 55 kD protein and the commercial monoclonal antiserum against type 7 cytokeratism), a single band was obtained in the 55 kD region. It is the only band detectable after SDS-PAGE and staining with Coomassie blue, recognized both by antι-55 kD sera and by commercial anti-mesothelial cytokeratin monoclonal antibodies. It is possible, therefore, that the 55 kD protein, possibly using its lectin functions, is able to bind to albumin, thereby giving SCL a false appearance of higher PM than it actually does.

This hypothesis is also supported by the fact that in highly purified commercial bovine albumin (99%), using the commercial monoclonal antiserum, a second band of a slightly weaker P.M. is detected. After treatment of the albumin preparation with pepsin, this band persists, while the albumin is digested (see FIGS. 4A and 4B). The relation between albumin and SCL could be purely coincidental, or on the contrary important for the diffusion of SCL in the organism, and for the maintenance of its stability.

3) In all the tests carried out, the majority of SCLs are found excreted in the culture medium, unlike the intermediate filaments which are generally intracellular. 4) Overall, the protein sequences obtained can be diagrammed as follows:

^{* •} )

B C

illet β helix α ^r illet β

The A domain corresponding to approximately 320 bp in DNA is variable; it is translated into a β 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 α 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 β sheet which also has lectin sequences.

5) 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 α-helix sequences stable to the molecule.

On the contrary, the invention establishes that the variable 5 ′ segment forming the β sheet contains the lectmic function of the molecule. The stable domain of the four α 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. In addition, the SCL of the invention, as noted above, 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 diméπque. The intermediate filaments are, on the contrary, polymerized into multimeres.

6) The household treatment with pepsin at pH 2 of the purified SCL destroys the stimulatory function of the SCL, without modifying either the migration or the size of the molecule in the SDS-PAGE gel. Likewise, the agglutinative power of cells is preserved. He is known in effect that pepsin first destroys sites localized at the 5 'end of the molecule (at the level of the aromatic ammo-acids threonme and tyrosme) explaining the loss of biological functions.

EXAMPLE 5: STUDY OF BIOLOGICAL ACTIVITY

I - Study of the inhibitory effects of sugars vis-

Action on simple sugars ^r aggl utina tion of cell ules.

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.

In the example presented, 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.

D galactose: refined 0.28 mM β lactose: 0.25 mM α lactose: 0.125 mM

N. A. N. A: 0.0075 mM

In the case of murine sarcolectin, the affinity is higher for β 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.

This effect will be all the more pronounced the greater the affinity for the SCL receptors.

We will therefore measure the interest of the SCLs of the invention which make it possible to select sugars with a very high inhibitory effect and from which we can take advantage of the indirect effect on growth. As already pointed out, this sugar-inhibiting effect with respect to SCLs makes it possible to increase the antiviral and antiproliferative functions of IFNs.

Indirect anti-viral effects of inhibiting sugars

The encephalomyocarditis virus (CME), which kills mice in almost all cases, after fairly varied but relatively short incubation periods, is used as a model. Male mice are injected (average weight:

2.5 g, 8 to 10 weeks old), via the sodium galacturonate route, glucosamine and N-acetyl-neuraminic acid (5 to 10% solutions containing 100 mM sugar). 24 hours later, one hundred lethal doses (50%) of EMC virus are injected by the same route. After one hour, the murine interferon is injected (at a dose of 20,000 units in 0.5 ml). Table 3 gives the numbers of animals surviving on the total tested, percentage and statistical significance.

Protection of IFN against the pathogenic effect of the vO -J virus

Encephalomyocarditis in Swiss O vβ mice

(Number of surviving animals out of total number tested, N> percentage and statistical significance [*])

4> ~ -J

"S fi

H ev

-α

Examination of these results shows that the use of SCL-inhibiting sugars makes it possible to reduce the antagonistic effect of natural sarcolectins, the rate of which increases due to immune stimulation due to the virus, resulting in increased synthesis of macrophages with T lymphocytes. .

In mice treated only with

1 interferon, only six animals out of 120 survive, while the increase in total survival is very clear when combined with interferon N.A.N.A., glucosamine or galacturonate.

By providing SCL constituting models for in vitro studies, 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.

Anti tumor action of SCL inhibitor sugars

The ability of inhibitory sugars to hinder the growth-stimulating effect induced by sarcolectin may indirectly affect tumor development in vivo. As demonstrated in vitro according to the invention, 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. To test the anti-tumor power of the SCL inhibitor sugars, 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.

In parallel series, the different groups of animals were treated 3 days after inoculation, either with a specific sugar

(glucosamine, lactose, galacturonate, NANA, neuramine-lactose), in solution from 5 to 10%, the sugar concentration being 100 mM, either by the same treatment to which an injection of immunostimulant (extract of Corynebacterium parvum, Mérieux, France) at a dose of

200μg / mice, or interferon, or both.

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

On the tenth day after the transplant, treatment with all the sugars tested, either alone or in combination with Corynebacterium parvum (CP) or IFN, considerably reduces the incidence of tumors. Average survival is particularly increased by glucosamine and galacturonate. In the case of a single immunostimulation by CP, total survival is considerably increased by glucosamine (53 °) and lactose (43 °). The association with IFN increases the average survival especially for lactose.

II- Study of the inhibitory effects of butyric amino acids with respect to SCLs

Indirect anti-tumor effects of butyric ammo-acids The same experimental procedure as for the preceding table is used, but using butyric salts: gamma ammo-butyric

(GABA), alpha ammo-butyric, alpha-ammo-isobutyric, and control medium. The results obtained are collated in Table 5:

TABLE 5

Amino acid Number STD tumors Survival from day 10 to day Number final mouse Number (%) (%)

GABA 30 14 (46) 35 + 9 4 (13)

+ IFN 45 19 (42) 43 + 10 11 (24)

+ CP 45 25 (55) 68 + 11 24 (53)

+ CP + IFN 30 7 (23) 70 + _ 13 17 (56)

alpha amino goal. 30 19 (63) 26 + _ 1 0 (0)

+ IFN 30 9 (30) 25 + 2 0 (0)

+ CP 30 18 (60) 55 + 14 7 (23)

+ CP + IFN 30 7 (23) 62 + 14 14 (47)

alpha amino-iso. 30 6 (20) 31 + 7 2 (7)

+ IFN 30 15 (50) 31 + 10 4 (13)

+ CP 30 14 (46) 66 + 13 13 (43)

+ CP + IFN 30 12 (40) 64 + _ 13 14 (47)

Middle 105 94 (89) 19 + 1 0 (0)

+ IFN 105 65 (68) 25 + 4 7 (7)

+ CP 105 85 (89) 32 + 5 10 (10)

+ CP + IFN 105 43 (41) 60 + 7 45 (42)

Under the same conditions as the sarcolectin-inhibiting sugars in the previous series, 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.

As in the case of inhibiting sugars, 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).

III- Direct effects of sarcolectin on the stimulation of cellular DNA synthesis

We verified the effect of sarcolectin on the DNA synthesis of different immunocompetent cells: 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. In all cases, the growth medium did not contain serum, only highly purified sarcolectin as obtained according to Example 1.

After 24 hours, the DNA synthesis of the different cell populations was analyzed by comparing the control cells treated with the same medium and without sarcolectin. There is a significant increase in DNA synthesis, especially for H9, Daudi and U937 cells, slightly less for normal T and B cells and HeLa cells, while it has remained at the same high level as non-anchored lines for mouse cells. These results clearly show that sarcolectin has its own stimulating effect on growth, which is independent of the more specific growth factors to which it must be added.

In the biopsies taken during the osteogenic sarcoma examinations in children, the fragment excretes in a serum-free medium considerable quantities of SCL identified by Western blot tests using antiserum antι-55 kD.

This same lectin is also found in the ascites removed during transplants of sarcomas TG 180 of the mouse (track 4, in FIG. 5), tracks 1 to 3 corresponding to non-cancer patients, and tracks 5 to 8 to human osteogenic sarcoma sera.

IV- Inhibitory effect of sarcolectin on the synthesis of secondary effector proteins of interferon

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. These data suggest that, without affecting its production, the action of interferon (induced by poly (I) (C)), can be inhibited by SCL, which results in the reduction or total disappearance of retromhibition. .

Under these conditions, the specific effect of poly (I) (C) on growth appears clearly. The IFN and the SCL are in equilibrium and also act on the poly (I) (C) by modifying its expression, the whole resulting in a triangular equilibrium.

V- Use of sarcolectin for diagnosis

As sarcolectin stimulates DNA synthesis and inhibits secondary effector proteins

1 Interferon, its presence can be sought both in tumors as in different biological secretions, serum, ascites liquids, placenta, etc.

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;

- lowering the pH of the medium to pH 5 for 30 mm at laboratory temperature;

- readjustment to pH 7.4; there is then a significant precipitation which must be eliminated by centrifugation at 10,000 revolutions / mm. for 30 mm. ;

- readjustment of the pH of the supernatant obtained to pH 7.4.

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.

VI- Search for sarcolectin by agglutination of cells

By way of example, it is possible to use 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.

VII- Analysis of sarcolectin by increasing the synthesis of cellular DNA

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.

VIII - Use of SCL in anti-tumor therapy In anti-tumor therapy, it is possible, as described below, to consider the use of SCL, provided that there is not beforehand constitutive production of SCL.

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.

According to Lampl et al. (11) growth spurts are short and brutal for 24 hours, followed by a 30-60 day stopping period. On these bases, the treatment offered is as follows:

1. Unique stimulation of growth by the SCL used by the parental way in sufficient doses to significantly increase the proliferation of leukocytes in the blood. This function can be amplified by aspartate salts (24 mM / kg.) Administered in parallel for the first 3 days. It can be replaced by cimetidme.

Recombinant interleukin 2 can also be combined under comparable conditions.

2. After stopping for 4-5 days, the target treatment includes IFNs, especially of the α or β 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.

3. The effect of interferons can be amplified by the combination of the sugars mentioned above, in particular lactoses (α or β), D galactose, N-acetyl neurammic acid (NANA) chosen as example. 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, α lactose, NANA

IX Reproduction of essential biological functions with recombinant SCL

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 results obtained are reported in Table 6.

It is found that in the presence of cells incubated with interferon for 5 hours, the antiviral function is expressed. From the fifth hour, we replaces interferon with SCL for the next 18 hours, taking the replacement of SCL with the medium in the control cell; the recombinant SCL blocks the state of the antiviral effect whereas in the controls treated with the medium, the interferon already fixed on the receptors expresses all of its functions normally.

In a second series of experiments, 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.

TABLE 6

INHIBITION OF INTERFERON ACTION (IFN) BY RECOMBINANT SCL.

A. IFN TITLE 32 U

concentration of SCL which inhibits 100% the antiviral state IX Induction of antibodies against oligopeptides SEQ ID N ° 5 or SEQ ID N ° 6

By operating according to conventional techniques, the formation of antibodies against the peptides SEQ ID No. 5 and SEQ ID No. 6 is induced. The specific characteristics of these antibodies are reported in Table 7.

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. XI Stimulation in 24 hours. of DNA synthesis in (lympho) T cells

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.

Examination of the results in Table 8 shows that the SCL excreted from the cells stimulates DNA synthesis in 24 h.

This stimulation is inhibited by NANA or by oligopeptide 41-55 (SEQ ID No. 5). BIBLIOGRAPHICAL REFERENCES

1 / Fournier F., et al, (1969), Proc. Soc. Exp.

Biol. Med. 132, 943

2 / Chany C, et al, (1971), Nature, Biol. 230, 11

3 / Fournier F. et al (1972) Nature New Biol., 274, 1757.

4 / Chany, C. et al, (1969), CR. Acad. Sci. Paris 269, 1236

5 / Duc-Goiran P. et al. (1985), Proc. Nat. Acad. Sci USA, 82.5010 6 / Chany C. et al (1969), 269.2628

7 / Jiang P.H. et al (1988) Biol., Chemistry, 263, 19154

8 / Chany-Fournier F. et al (1990). I of Cellular Physiology, 145, 173 9 / Glass C. et al, (1985) J. Cell Biol. 101,

236

10 / Sambrook J. et al, (1989) Cold Spring Harbor Laboratory Press

11 / Lampl M et al (1992) Science 258; 801 12 / Fu-Yue Zeng et al (1994) Bio Chem 375, 393-

399.

LIST OF SEQUENCES

11) GENERAL INFORMATION:

(l) DEPOSITOR:

(A) NAME: A.D.B.E.A

ASSOCIATION FOR THE DEVELOPMENT OF EXPERIMENTAL AND APPLIED BIOTHERAPY HOSPITAL SAINT VINCENT DE PAUL

(B) STREET: 74-82 AVENUE DENFERT ROCHEREAU

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75674

(II) TITLE OF THE INVENTION: LECTIN-LIKE PROTEINS AND THEIR BIOLOGICAL APPLICATIONS

(m) NUMBER OF SEQUENCES: 6

(iv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 1831 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (111) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(îx) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 62..1469

(Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

GAATTCCGGC GAGTGCGCGC TCCTCCTCGC CCGCCGCTAG GTCCATCCCG GCCCAGCCAC 60

C ATG TCC ATC CAC TTC AGC TCC CCG GTA TTC ACC TCG CGC TCA GCC 106

Met Ser Ile His Phe Ser Ser Pro Val Phe Thr Ser Arg Ser Ala 1 5 10 15

GCC TTC TCG GGC CGC GGC GCC CAG GTG CGC CTG AGC TCC GCT CGC CCC 154 Ala Phe Ser Gly Arg Gly Ala Gin Val Arg Leu Ser Ser Ala Arg Pro 20 25 30

GGC GGC CTT GGC AGC AGC AGC CTC TAC GGC CTC GGC GCC TCG CGG CCG 202 Gly Gly Leu Gly Ser Ser Ser Leu Tyr Gly Leu Gly Ala Ser Arg Pro 35 40 45

CGC GTG GCC GTG CGC TCT GCC TAT GGG GGC CCG GTG GGC GCC GGC ATC 250 Arg Val Ala Val Arg Ser Ala Tvr Gly Gly Pro Val Gly Ala Gly Ile 50 55 60 CGC GAG GTC ACC ATT AAC CAG AGC CTG CTG GCC CCG CTG CGG CTG GGC 298 Arg Glu Val Thr Ile Asn Gin Ser Leu Leu Ala Pro Leu Arg Leu Gly 65 70 75

GCC GAC CCC TTC TCC CAG CGG GTG CGC CAG GAG GAG AGC GAG CAG ATC 346 Ala Asp Pro Phe Ser Gin Arg Val Arg Gin Glu Glu Ser Glu Gin Ile 80 85 90 95

AAG ACC CTC AAC AAC AAG TTT GCC TCC TTC ATC GAC AAG GTG CGG TTT 394 Lys Thr Leu Asn Asn Lys Phe Ala Ser Phe Ile Asp Lys Val Arg Phe 100 105 110

CTG GAG CAG CAG AAC AAG CTG CTG GAG ACC AAG TGG ACG CTG CTG CAG 442 Leu Glu Gin Gin Asn Lys Leu Leu Glu Thr Lys Trp Thr Leu Leu Gin 115 120 125

GAG CAG AAG TCG GCC AAG AGC AGC CGC CTC CCA GAC ATC TTT GAG GCC 490 Glu Gin Lys Ser Ala Lys Ser Ser Arg Leu Pro Asp Ile Phe Glu Ala 130 135 140

CAG ATT GCT GGC CTT CGG GGT CAG CTT GAG GCA ATG CAG GTG GAT GGG 538 Gin Ile Ala Gly Leu Arg Gly Gin Leu Glu Ala Met Gin Val ASD Gly 145 150 155

GGC CGC CTG GAG CAG GGG CTG CGG ACG ATG CAG GAT GTG GTG GAG GAC 586 Gly Arg Leu Glu Gin Gly Leu Arg Thr Met Gin Asp Val Val Glu Asp 160 165 170 175

TTC AAG AAT AAG TAC GAA GAT GAA ATT AAC CGC CGC ACA GCT GCT GAG 634 Phe Lys Asn Lys Tyr Glu Asp Glu Ile Asn Arg Arg Thr Ala Ala Glu 180 185 190

AAT GAG TTT GTG GTC CTG AAG AAG GAT GTG GAT GCT GCC TAC ATG AGC 682 Asn Glu Phe Val Val Leu Lys Lys Asp Val Asp Ala Ala Tyr Met Ser 195 200 205

AAG GTG GAG CTG GAG GCC AAG GTG GAT GCC CTG AAT GAT GAG ATC AAC 730 Lys Val Glu Leu Glu Ala Lys Val Asp Ala Leu Asn Asp Glu Ile Asn 210 215 220

TTC CTC AGG ACC CTC AAT GAG ACG GAG TTG ACA GAG CTT CAG TCC CAG 778 Phe Leu Arg Thr Leu Asn Glu Thr Glu Leu Thr Glu Leu Gin Ser Gin 225 230 235

ATC TCC GAC ACA TCT GTG GTG CTG TCC ATG GAC AAC AGT CGC TCC CTG 826 Ile Ser Asp Thr Ser Val Val Leu Ser Met Asp Asn Ser Arg Ser Leu 240 245 250 255

GAC CTG GAC GGC ATC ATC GCT GAG GTC AAG GCG CAG TAT GAG GAG ATG 874 Asp Leu Asp Gly Ile Ala Glu Val Lys Ala Gin Tyr Glu Glu Met 260 265 270

GCC AAA TGC AGC CGG GCT GAG GCT GAA GCC TGG TAC CAG ACC AAG TTT 922 Ala Lys Cys Ser Arg Ala Glu Ala Glu Ala Trp Tyr Gin Thr Lys Phe 275 280 285

GAG ACC CTC CAG GCC CAG GCT GGG AAG CAT GGG GAC GAC CTC CGG AAT 970 Glu Thr Leu Gin Ala Gin Ala Gly Lys His Gly Asp Asp Leu Arg Asn 290 295 300

ACC CGG AAT GAG ATT TCA GAG ATG AAC CGG GCC ATC CAG AGG CTG CAG 1018 Thr Arg Asn Glu Ile Ser Glu Met Asn Arg Ala Ile Gin Arg Leu Gin 305 310 315

GCT GAG ATC GAC AAC ATC AAG AAC CAG CGT GCC AAG TTG GAG GCC GCC 1066 Ala Glu Ile Asp Asn Ile Lys Asn Gin Arg Ala Lys Leu Glu Ala Ala 320 325 330 335 ATT GCC GAG GCT GAG GAG CGT GGG GAG CTG GCG CTC AAG GAT GCT CGT 1114 Ile Ala Glu Ala Glu Glu Arg Gly Glu Leu Ala Leu Lys Asp Ala Arg 340 345 350

GCC AAG CAG GAG GAG CTT GAA GCC GCC CTG CAG CGG GCC AAG CAG GAT 1162 Ala Lys Gin Glu Glu Leu Glu Ala Ala Leu Gin Arg Ala Lys Gin Asp 355 360 365

ATG GCA CGG CAG CTG CGT GAG TAC CAG GAA CTC ATG AGC GTG AAG CTG 1210 Met Ala Arg Gin Leu Arg Glu Tyr Gin Glu Leu Met Ser Val Lys Leu 370 375 380

GCC CTG GAC ATC GAG ATC GCC ACC TAC CGC AAG CTG CTG GAG GGC GAG 1258 Ala Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu 385 390 395

GAG AGC CGG TTG GCT GGA GAT GGA GTG GGA GCC GCC AAT ATC TCT GTG 1306 Glu Ser Arg Leu Ala Gly Asp Gly Val Gly Ala Ala Asn Ile Ser Val 400 405 410 415

ATG AAT TCC ACT GGT GGC AGC AGC AGT GGC GGT GGC ATT GGG CTG ACC 1354 Met Asn Ser Thr Gly Gly Ser Ser Ser Gly Gly Gly Ile Gly Leu Thr 420 425 430

CTC GGG GGA ACC ATG GGC AGC AAT GCC CTG AGC TTC TCC AGC AGT GCG 1402 Leu Gly Gly Thr Met Gly Ser Asn Ala Leu Ser Phe Ser Ser Ser Ala 435 440 445

GGT CCT GGG CTC CTG AAG GCT TAT TCC ATC CGG ACC GCA TCC GCC AGT 1450 Gly Pro Gly Leu Leu Lys Ala Tyr Ser Ile Arg Thr Ala Ser Ala Ser 450 455 460

CGC AGG AGT ACC CGC GAC TGAGTCGC CTCCCACCAC TCCACTCCTC 1496

Arg Arg Ser Thr Arg Asp 465

CAGCCACCAC CCACAATCAC AGCCATTGCC GAGGCTGAGG AGTGTGGGGA GCTGGCGCTC 1556

AAGGATGCTC GTGCCAAGCA GGAGGAGCTG GAAGCCGCCC TGCAGCGGGC CAAGCAGGAT 1616

ATGGCACGGC AGCTGCGTGA GTACCAGGAA CTCATGAGCG TGAAGCTGGC CCTGGACATC 1676

GAGATCGCCA CCTACCGCAA GCTGCTGGAG GGCGAGGAGA GCCGGTTGGC TGGAGATGGA 1736

GTGGGAGCCG TGAATATCTC TGTGATGAAT TCCACTGGTG GCAGTAGCAG TGGCGGTGGC 1796

ATTGGGCTAG CCCTCGGGGG AACCATGGGC AGCAA 1831

(3) INFORMATION FOR SEQ ID NO: 2:

(1) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 405 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(îx) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION:! .. 405

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: ATG TCC ATC CAC TTC AGC TCC CCG GTA TTC ACC TCG CGC TCA GCC GCC Met Ser Ile His Phe Ser Ser Pro Val Phe Thr Ser Arg Ser Ala Ala 1 5 10 15

TTC TCG GGC CGC GGC GCC CAG GTG CGC CTG AGC TCC GCT CGC CCC GGC 96 Phe Ser Glv Arg Gly Ala Gin Val Arg Leu Ser Ser Ala Arg Pro Gly 20 25 30

GGC CTT GGC AGC AGC AGC CTC TAC GGC CTC GGC GCC TCG CGG CCG CGC 144 Gly Leu Glv Ser Ser Ser Leu Tyr Gly Leu Gly Ala Ser Arg Pro Arg 35 40 45

GTG GCC GTG CGC TCT GCC TAT GGG GGC CCG GTG GGC GCC GGC ATC CGC 192 Val Ala Val Arg Ser Ala Tyr Gly Gly Pro Val Gly Ala Gly Ile Arg 50 55 60

GAG GTC ACC ATT AAC CAG AGC CTG CTG GCC CCG CTG CGG CTG GGC GCC 240 Glu Val Thr Ile Asn Gin Ser Leu Leu Ala Pro Leu Arg Leu Gly Ala 65 70 75 80

GAC CCC TTC TCC CAG CGG GTG CGC CAG GAG GAG AGC GAG CAG ATC AAG 288 Asp Pro Phe Ser Gin Arg Val Arg Gin Glu Glu Ser Glu Gin Ile Lys 85 90 95

ACC CTC AAC AAC AAG TTT GCC TCC TTC ATC GAC AAG GTG CGG TTT CTG 336 Thr Leu Asn Asn Lys Phe Ala Ser Phe Ile Asp Lys Val Arg Phe Leu 100 105 110

GAG CAG CAG AAC AAG CTG CTG GAG ACC AAG TGG ACG CTG CTG CAG GAG 384 Glu Gin Gin Asn Lys Leu Leu Glu Thr Lys Trp Thr Leu Leu Gin Glu 115 120 125

CAG AAG TCG GCC AAG AGC AGC 405

Gin Lys Ser Ala Lys Ser Ser 130 135

(4) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 469 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: protein

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

Met Ser Ile His Phe Ser Ser Pro Val Phe Thr Ser Arg Ser Ala Ala 1 5 10 15

Phe Ser Gly Arg Gly Ala Gin Val Arg Leu Ser Ser Ala Arg Pro Gly 20 25 30

Gly Leu Gly Ser Ser Ser Leu Tyr Gly Leu Gly Ala Ser Arg Pro Arg 35 40 45

Val Ala Val Arg Ser Ala Tyr Gly Gly Pro Val Gly Ala Gly Ile Arg 50 55 60

Glu Val Thr Ile Asn Gin Ser Leu Leu Ala Pro Leu Arg Leu Gly Ala 65 70 75 80

Asp Pro Phe Ser Gin Arg Val Arg Gin Glu Glu Ser Glu Gin Ile Lys 85 90 95

Thr Leu Asn Asn Lys Phe Ala Ser Phe Ile Asp Lys Val Arg Phe Leu 100 105 110

Glu Gin Gin Asn Lys Leu Leu Glu Thr Lys Trp Thr Leu Leu Gin Glu 115 120 125 Gin Lys Ser Ala Lys Ser Ser Arg Leu Pro Asp Ile Phe Glu Ala Gin 130 135 140

Ile Ala Gly Leu Arg Gly Gin Leu Glu Ala Met Gin Val Asp Gly Gly 145 150 155 160

Arg Leu Glu Gin Gly Leu Arg Thr Met Gin Asp Val Val Glu Asp Phe 165 170 175

Lys Asn Lys Tyr Glu Asp Glu Ile Asn Arg Arg Thr Ala Ala Glu Asn 180 185 190

Glu Phe Val Val Leu Lys Lys Asp Val Asp Ala Ala Tyr Met Ser Lys 195 200 205

Val Glu Leu Glu Ala Lys Val Asp Ala Leu Asn Asp Glu Ile Asn Phe 210 215 220

Leu Arg Thr Leu Asn Glu Thr Glu Leu Thr Glu Leu Gin Ser Gin Ile 225 230 235 240

Ser Asp Thr Ser Val Val Leu Ser Met Asp Asn Ser Arg Ser Leu Asp 245 250 255

Leu Asp Gly Ile Ile Ala Glu Val Lys Ala Gin Tyr Glu Glu Met Ala 260 265 270

Lys Cys Ser Arg Ala Glu Ala Glu Ala Trp Tyr Gin Thr Lys Phe Glu 275 280 285

Thr Leu Gin Ala Gin Ala Gly Lys His Gly Asp Asp Leu Arg Asn Thr 290 295 300

Arg Asn Glu Ile Ser Glu Met Asn Arg Ala Ile Gin Arg Leu Gin Ala 305 310 315 320

Glu Ile Asp Asn Ile Lys Asn Gin Arg Ala Lys Leu Glu Ala Ala Ile 325 330 335

Ala Glu Ala Glu Glu Arg Gly Glu Leu Ala Leu Lys Asp Ala Arg Ala 340 345 350

Lys Gin Glu Glu Leu Glu Ala Ala Leu Gin Arg Ala Lys Gin Asp Met 355 360 365

Ala Arg Gin Leu Arg Glu Tyr Gin Glu Leu Met Ser Val Lys Leu Ala 370 375 380

Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu 385 390 395 400

Ser Arg Leu Ala Gly Asp Gly Val Gly Ala Ala Asn Ile Ser Val Met 405 410 415

Asn Ser Thr Gly Gly Ser Ser Gly Gly Gly Ile Gly Leu Thr Leu 420 425 430

Gly Gly Thr Met Gly Ser Asn Ala Leu Ser Phe Ser Ser Ser Ala Gly 435 440 445

Pro Gly Leu Leu Lys Ala Tyr Ser Ile Arg Thr Ala Ser Ala Ser Arg 450 455 460

Arg Ser Thr Arg Asp 465 (5) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 135 amino acids

(B) TYPE: amino acid

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: protein

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

Met Ser Ile His Phe Ser Ser Pro Val Phe Thr Ser Arg Ser Ala Ala

1 5 10 15

Phe Ser Gly Arg Gly Ala Gin Val Arg Leu Ser Ser Ala Arg Pro Gly 20 25 30

Gly Leu Gly Ser Ser Ser Leu Tyr Gly Leu Gly Ala Ser Arg Pro Arg 35 40 45

Val Ala Val Arg Ser Ala Tyr Gly Gly Pro Val Gly Ala Gly Ile Arg 50 55 60

Glu Val Thr Ile Asn Gin Ser Leu Leu Ala Pro Leu Arg Leu Gly Ala

65 70 75 80

Asp Pro Phe Ser Gin Arg Val Arg Gin Glu Glu Ser Glu Gin Ile Lys 85 90 95

Thr Leu Asn Asn Lys Phe Ala Ser Phe Ile Asp Lys Val Arg Phe Leu 100 105 110

Glu Gin Gin Asn Lys Leu Leu Glu Thr Lys Trp Thr Leu Leu Gin Glu 115 120 125

Gin Lys Ser Ala Lys Ser Ser 130 135

(6) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) NUMBER OF STRANDS:

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: peptide (m) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Gly Leu Gly Ala Ser Arg Pro Arg Val Ala Val Arg Ser Ala Tyr 1 5 10 15 (7) INFORMATION FOR SEQ ID NO: 6:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) NUMBER OF STRANDS:

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Asp Pro Phe Ser Gin Arg Val Arg Gin Glu Glu Ser Glu Gin Ile

1 5 10 15

Claims

1 / Nucleotide sequences, characterized in that they are isolated from their natural environment and 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 encoding polypeptides with lectinic properties. 2 / Nucleotide sequences, characterized in that they are isolated from their natural environment and that they are capable of coding, implemented according to conventional techniques of recombinant DNA, for sarcolectins comprising at least one sequence of amino acids as indicated in SEQ ID No. 1, in which one or more amino acids are modified, if necessary.

3 / Nucleotide sequences, characterized in that they are isolated from their natural environment and that they are capable of hybridizing with at least one fragment of SEQ ID No 1 carrying at least part of the genetic information for a sarcolectin.

4 / Nucleotide sequences according to one of claims 1 to 3, characterized in that it corresponds to the open reading frame of 1407 bp going from position 62, in SEQ ID No. 1, to position 1469.

5 / Nucleotide sequences according to one of claims 1 to 4, characterized in that they comprise at least part of the 5 'region of SEQ ID No 1 and / or of the 3' region.

6 / Nucleotide sequences according to claim 5, characterized in that they comprise at least part of the sequence SEQ ID No. 2, one or more nucleotides being modified if necessary.

7 / The mRNA sequences corresponding to the sequences according to one of claims 1 to 6. 8 / The antisense sequences corresponding to the sequences according to one of the preceding claims.

9 / The cDNAs corresponding to the sequences according to one of claims 1 to 8.

10 / Expression vectors containing at least one of the nucleotide sequences according to one of the preceding claims, subjected to the control of an appropriate promoter.

11 / Host cells transfected with the vectors according to claim 10, comprising the recombinant proteins expressed.

12 / New compounds, characterized in that they have lectmic activity and comprise at least part of a sequence of amine acids in the sequence coded by one of the nucleotide sequences defined in one of claims 1 a 6, one or more amine acids being modified if necessary, provided that this modification does not alter the lectin properties of sarcolectins.

13 / New compounds, characterized in that they have a sequence of amine acids in SEQ ID No. 3 or SEQ ID No. 4.

14 / New compounds, characterized in that they correspond to the sequence coded by the open reading frame of SEQ ID No. 1 and include 469 amine acids, their molecular weight being evaluated at approximately 55kD. 15 / New compounds, 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 in one of claims 1 to 6, recovery of the expressed compound and purification.

16 / New compounds, characterized in that they are fragments or derivatives of the compounds according to one of claims 12 to 15, since they retain at least part of the lectin properties.

17 / New compounds according to claim

16, characterized in that they are peptides SEQ ID No. 5.

18 / New compounds according to claim 17, characterized in that they are peptides having the sequence of the fragment 41 to 55 in SEQ ID No. 1, or peptides derived from this sequence and 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.

19 / New compounds according to claim 16, characterized in that they are peptides SEQ ID No. 6. 20 / New compounds according to claim 17, characterized in that they are peptides having the sequence of the fragment 81 to 95 in SEQ ID No. 1, or peptides derived from this sequence and 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 41 to 55 in SEQ ID No. 1.

21 / Compounds according to claim 12, as obtained by purification from tissue extracts, by a process comprising: the treatment of the tissue extract containing lectins, under conditions provided with pepsin or at acid pH, under conditions making it possible to eliminate at least the majority of the contaminating proteins while retaining lectin activity,

- chromatography on Sephacryl-S-200 and, if necessary, DEAE cellulose

- chromatography on CM-Trisacryl-M - affinity chromatography using a sugar such as N-acetyl neuraminic acid as ligand, and

- separation in reverse HPLC,

22 / Compounds according to claim 21, characterized in that the fraction separated in reverse HPLC is subjected to electrophoresis on SDS-PAGE gel and denaturation and that the bands corresponding to 55kD and £ 14kD are recovered.

23 / The antibodies directed against the compounds according to one of claims 12 to 22.

24 / Antibodies according to claim 23, characterized in that they are antibodies directed against the 55 kD protein according to claim 22 and that they recognize, in addition to the 55 kD protein, also the 65 kD protein. 25 / A method of obtaining compounds according to claim 12 or 21, a high degree of purity comprising the treatment of a tissue extract containing sarcolectin with pepsin or at acidic pH so as to eliminate at least the ma my part contaminating proteins of the extract while retaining biological lectmic activity, and

1 elimination of impurities by chromatography on

Sephacryl-S -200 and DEAE cellulose, characterized in that it further comprises a chromatography step on CM-Trisacryl-M, then affinity chromatography using as ligand N-acetyl-neurammic acid, purity SCLs are verified by HPLC if desired. 26 / A method according to claim 25, characterized in that the chromatography step on CM-Tnsacryl-M is carried out using a first buffer in order to remove at least the majority of the contaminating albumin, then d 'a second buffer to elute the SCL retained on the column.

27 / A method according to claim 25 or 26, characterized in that the affinity chromatography step carried out with a sugar as a ligand comprises the use of an agarose gel column on which the sugar is fixed and at least two buffers, so as to elute the SCL first, then to remove the contaminating proteins.

28 / A method according to one of claims 25 to 27, characterized in that the separation in HPLC is carried out using a water / acetonitrile / trifluoroacetic acid system.

29 / A method according to claim 28, characterized in that the fraction corresponding to the peak main in the HPLC step is subjected to an electrophoresis on SDS-PAGE gel and denaturation and that the bands of 65kD, 55 kD and £ 14 kD are recovered respectively. 30 / Process for obtaining sarcolectins with a high degree of purity, characterized in that it comprises:

- lowering the pH of the medium to pH 5 for 30 minutes;

- lowering to pH 5 which leads to a large precipitate, which will be removed by centrifugation;

- readjustment to pH 7.4 and recovery of the supernatant which contains the proteins 65 and 55 kD recognizable by the Western blots using specific antibodies.

31 / Growth factors, which can be used more particularly to contribute to the regeneration of damaged tissues and to the improvement of healing processes, characterized in that it is SCL according to one of claims 12 to 22.

32 / Therapeutic agents for stimulating the immune system, in particular specific immunity, characterized in that it is SCL according to one of claims 12 to 22, where appropriate in combination with specific growth factors , like interleukin 2.

33 / Use of the SCLs according to one of claims 12 to 22, for selecting inhibitors of their lectmic activity, such as sugars, aminobutyric acids and optionally high dose interferons. 34 / Agents capable of blocking the action of

SCL produced in excess in pathological conditions, such as cancer, chronic viral or autoimmune diseases, characterized in that they are antibodies according to one of claims 23 or 24.

35 / In vitro method of biological determination of

SCL, characterized in that it comprises bringing a biological sample to be analyzed or cells into contact with an anti-SCL antibody preparation according to claim

21, or an antibody fragment, the antibody used being immobilized on a solid support, under conditions suitable for the production of an antigen-antibody complex, with the SCLs when they are present in the sample or cells,

- highlighting the formation of such an antigen-antibody type complex.

36 / Kit for the diagnosis of the presence of SCL in a biological sample or cells, characterized in that it comprises an appropriate solid phase serving as a support,

- A preparation of anti-SCL antibodies according to claim 24 or of fragments, free or immobilized, directed against peptides 41-55.

- buffer solutions and reagents suitable for immunological reactions and for detection reactions.

37 / Method for detecting SCL in vitro, characterized in that it brings a biological sample to be analyzed or cells into contact with a probe produced from a nucleotide fragment according to one of claims 1 to 7 or 9, under conditions suitable for the production of a hybridization complex with DNAs coding for SCLs when they are present in the sample or cells, and

- highlighting the formation of the hybridization complex when the SCLs are present in the sample or the cells.

38 / Kit for in-vitro detection of the presence of genes coding for SCLs, characterized in that it comprises:

- nucleotide probes produced from the sequences defined in one of claims 1 to 7 or 9, and - buffer solutions and reagents for the hybridization reaction.

39 / Use of the antisense sequences according to claim 8, to block the expression of SCL according to one of claims 12 to 22. 40 / Use of a compound according to one of claims 12 to 20 attached to l albumin to make a delay vehicle.

41 / Use of a compound according to any one of claims 12 to 22, in particular of human recombinant products, as a growth factor for cells in vitro.