Soluble polypeptide fraction protein lag-3, a method of obtaining a therapeutic composition, the antibody


(57) Abstract:

The invention relates to medicine and biotechnology and includes soluble polypeptide fraction protein LAG-3, a method of obtaining a therapeutic composition and the antibody. The invention includes a soluble polypeptide fractions of membrane protein LAG-3, suitable for use as immunosuppressants, the method of production thereof, and antibodies capable of preventing specific fixation protein LAG-3 molecules SMS (major histocompatibility complex) class II, for use as Immunostimulants. The advantage of the invention is that the inventive peptides have the ability to bind with molecules SMS of the second class and have immunosuppressive effects, as well as unexpected immuno-stimulant, anti-CD4 antibodies. 6 C. and 8 C. p. F.-ly, 15 ill. , 5 PL.

The invention relates to soluble forms, derived from membrane protein LAG-3 and suitable as immunodepression, as well as antibodies capable of preventing specific fixation protein LAG-3 CMH molecules (major histocompatibility complex) class II, as Immunostimulants.

the active expressed by activated NK cells and activated T-lymphocytes. The similarity of amino acid sequence, comparative exon-intron structure and chromosomal localization show that LAG-3 is related CD 4. Initial characterization of the gene LAG-3 describes TRIEBEL and other (I).

The corresponding DNA encodes a transmembrane protein of 498 amino acids of type I, including 4 extracellular immunoglobulin sequence type. LAG-3 is a member of the superfamily of immunoglobulins.

The Mature protein consists of 476 amino acids (sequence No. 1) with theoretical molecular weight of 52 KD. The extracellular region contains 8 cysteine residues and 4 potential N-glycosylation. Using Western blotting analysis shows that LAG-3 in PHA blasts or activated NK cells has an apparent mass of Mr = 70000. After treatment with N-glycosidase - achieve size reduction of up to 60 KD, demonstrating that native LAG-3 glycolysation. The details are described in WIPO And 91/10682.

BAIXERAS, etc. in J. Exp. Med, 176, 327-337 (2), in addition, describe the formation of rosettes between transfitsirovannykh using LAG-3 cells (expressing LAG-3 on their surface) and B-lymphocytes expressing class II CMH, specifically depends on the EOI is more high quantities by activated CD8 lymphocytes (limited CMH class I), than on activated CD4 lymphocytes. In vivo, some single scattered cells LAG-3 (limited class II CMH) has been found in lymphoid tissues, including primary limfoidnye bodies, i.e. the thymus and bone marrow.

Cells LAG-3 is found in lymphoid giperplazicheskih nodes and tonsils, as well as in mononuclear cells of peripheral blood (PBMC) of patients receiving injections of high doses of interleukin-2.

These observations confirm that LAG-3 is an activation antigen in contrast to CD4 expressed in subpopulations of resting lymphocytes and other cell types, particularly in macrophages.

CMH includes molecules of class I and class II, which are membrane glycoproteins, which are fragments of protein antigens to receptors on T-lymphocytes (TCR). The class I molecules responsible for the presentation of cytotoxic cells CD8+peptides originating mostly from synthesized by endogenous proteins, whereas class II molecules are helper lymphocytes CD4+peptides originating primarily from foreign proteins that enter endianism, i.e. exogenous, which the ITA is required for cell disruption, whatever tissue expressing the antigen alien ("not his" type, for example, viral antigens. The detection mechanism is actuated by intracellular signals leading to efficient activation of T-lymphocytes.

It turns out that for the initiation of Immunotech on the mediation of T-lymphocytes (CD4+foreign antigens must be "captured", internality in the form of peptides specialized cells: antigen presenting cells (APC). The resulting antigenic peptides reexpression on the surface of antigen presenting cells, where they are associated with CMH molecules of the second class. This complex CMH second class / peptide specifically recognized by T-limfotsitam receptor, where does the activation of helper T-lymphocytes.

In addition, created ways recombination animal models allow us to emphasize the role that they play in vivo molecules CMH second class and their ligands.

Thus, mice that lack of CMH molecules of the second class (3) and virtually no peripheral T-lymphocytes CD4+and containing only a few immature lymphocytes CD4+in the thymus, completely unable to respond to T-dependent antigens.

+that demonstrates that the expression of CD4 in the daughter cells and thymocytes CD4+CD8+is not mandatory for development. Compared with normal mice, mice that lack CD4 have a greater number of CD4 cells-CD8-.

These cells with a double negative charge limited CMH second class and are able to recognize the antigen.

When they infect with Leishmania, these mice show the population of the auxiliary functional T-lymphocytes despite the absence of CD4. These cells are limited CMH second class and produce interferon when they are activated by antigen. This indicates that the origin of the T-lymphocytes and their peripheral function does not have to depend on the function of CD4.

Now consider that the coded plot CMH second class proteins are involved in many aspects of immune recognition, including there the interaction between different lymphoid cells as lymphocytes and antigen cells. Various observations also indicate that other mechanisms, not through CD4, participate in the activation function helper T-lymphocytes.

These various nablyudeniya, you know the part chimeric molecules formed unaryoperation domain proteins are able to bind to the ligands, and the constant region of chains of human immunoglobulins (Ig) in obtaining soluble proteins and cell receptors, especially suitable as therapeutic agents.

Thus, a soluble form of CD4 demonstrate its effectiveness in the inhibition of infection due to HIV in vitro dependent on dose.

However, clinical trials with soluble CD4 molecules, especially CD4 - Ig, failed to reveal a significant reduction in viral titers. Were created transgenic mice expressing up to 20 µg/ml of soluble CD4 in their serum. These mice show no differences in their immune function, compared with the control mice. Up to the present time there is no direct connection with CMH second class derived from CD4 molecules. This suggests that soluble CD4 do not interact in vivo with colored molecules CMH second class.

Unexpectedly, the authors of the present invention have found that soluble molecules containing different fragments unaryoperation domain FR">

Unaryoperation plot of LAG-3, denoted by the sequence N 1, includes the domains D1, D2, D3, D4, covering amino acids 1-149, 150-239, 240-330 and 331-412, respectively.

The subject invention is also soluble polypeptide (part) fraction formed in whole or in part, at least one of the four extracellular immunoglobulin domains of the type of protein LAG-3 (amino acids 1-149, 150 - 239, 240-330 and 331-412 sequence N 1) or a peptide sequence derived from these domains due to the replacement, addition and/or deletion of one or several amino acids, and which has a specificity of at least equal to or higher than that of the LAG-3 in its ligand.

The present invention particularly relates to a soluble polypeptide fractions having the sequence formed from the native sequence LAG-3 due to the well known phenomenon of policije.

Soluble polypeptide fraction differs in that it includes a peptide plot of LAG-3, responsible for the affinity of LAG-3 to the CMH molecules of the second class.

Soluble polypeptide fraction in particular includes the peptide sequence, formed from these domains, su, equal to or higher than that of the LAG-3 in its ligand, for example, the combination of the first two domains of the immunoglobulin type LAG-3 or 4 domain immunoglobulin type unaryoperation plot of LAG-3.

Preferably, the soluble polypeptide fraction formed in whole or in part, at least one of the four extracellular immunoglobulin domains of the type of protein LAG-3 (amino acids 1-149, 150-239, 240-330 and 331-412 sequence N 1), comprising the substitution of glutamic acid (Glu) of one or more arginine residues (Arg) at positions 73, 75 and 76 of the sequence N 1.

Preferably, the soluble polypeptide fraction includes a loop, the mean position of the atoms which constitute the main chain, represented by the position of amino acids 46-47 (sequence No. 1), are presented in table 1 or table 2, or differs from it by at most 5%.

Soluble polypeptide fraction, moreover, preferably includes the second extracellular domain (D2) immunoglobulin type LAG-3 (amino acids 150 -239).

Preferably, the soluble polypeptide fraction includes, in addition to the above peptide sequence LAG-3, additional peptide posledovatelnostyu, allows to modify the physico-chemical characteristics of subfragment unaryoperation domain protein LAG-3. Such fused proteins, for example, contain fragments unaryoperation domain of LAG-3, such as those mentioned above, associated with the region of the binding-CH2-CH3the heavy chain of human immunoglobulin, preferably immunoglobulin isotype IgG4.

Such fused proteins can be dimeric or Monomeric. These fused proteins can be obtained using well-known specialist methods of recombination, for example, using the method described by Traunecker and others (5).

In General, the method of obtaining these fused proteins, including immunoglobulin region, connected with the above peptide sequence LAG-3, is that in the vector-insert cDNA fragments encoding the polypeptide regions corresponding to the LAG-3 or a derivative LAG-3, possibly after amplification using the polymerase chain reaction (PCR) and cDNA encoding a suitable region of the immunoglobulin connected with cDNA that encodes a corresponding polypeptide or formed from LAG-3 region, and after transfection with the cDNA fragments Express in the expression system, especially the train can be obtained by splitting conjugate LAG-3 - - Ig is designed so that it contains the appropriate cleavage site.

The subject of the invention is a therapeutic composition with immunosuppressive activity, including soluble polypeptide fraction according to the invention. This composition suitable for the treatment of pathologies requiring immunosuppression, for example, autoimmune diseases.

The subject invention also is the use of antibodies directed against LAG-3, or soluble polypeptide fractions formed from LAG-3, such as those described above, or fragments of such antibodies, particularly fragments Fab, Fab', F(ab')2for the preparation of therapeutic compositions with immunostimulatory activity. The term "immunity" refers to a molecular complex, is able to stimulate the maturation, differentiation, proliferation and/or function of cells expressing LAG-3, that is, activated T-lymphocytes or NK-cells (natural killer cells). Antibodies against LAG-3 (anti-LAG-3 antibodies) can be used as potentialization (amps) vaccine or Immunostimulants in patients with depressed (depressed) by the immune system, such as the pain is stimulation of the immune system due to the removal of her cells, possessing abnormal condition such as cancer cells.

Immunostimulirutuyu activity anti-LG-3 antibodies is unexpected, where anti-CD4 antibodies exert immunosuppressive effects.

Such antibodies can be polyclonal or monoclonal; however, preferred monoclonal antibodies. Polyclonal antibodies can be obtained by well known methods, such as described BENEDICT, A. A., and others (6). Preferred monoclonal antibodies because they are specific for a single epitope and produce results with better reproducibility. Methods of producing monoclonal antibodies are well known in the prior art, in particular the method described by KOHLER and MILSTEIN. This method, as well as its variants described YELTON and others (7).

The subject invention are also antiidiotypic antibodies directed against the antibodies according to the invention, which include an "internal image" LAG-3 and, therefore, able to connect with CMH second class. Such antibodies can be used especially as immunosuppressive means and, for example, in autoimmune pathologies.

Therapeutic compositions according to the present IECI acceptable excipient. These compositions can be formulated in the usual way. Excipient may vary depending on the chosen route of administration: oral, parenteral, sublingual, rectal or through the nose.

If intended for parenteral administration of the compositions excipient usually includes sterile water, as well as other possible ingredients that increase the solubility or the suitability for storage of the composition. Parenteral routes of administration may be an intravenous, intramuscular or subcutaneous injection.

therapeutic composition can be prolonged action, especially for treatments over a long period of time, for example, in the case of autoimmune diseases. Enter the dosage depends on the treated subject, especially on the ability of the immune system to achieve the desired degree of protection. The exact number of the input of the active ingredient can be easily determined by a medical practitioner who prescribes a treatment.

Therapeutic compositions according to the invention can comprise, in addition to soluble LAG-3 or antibodies according to the invention, the other active ingredient, possibly associated with chemical communication with LAG-3 or with Anya, associated with toxin: such as ricin or diphtheria toxoid, is able to bind to the molecules CMH second class and kill cells against which they are directed, for example, leukemic cells or melanoma cells; or associated with a radioactive isotope.

The following examples, as well as standardized attached drawings illustrate the invention in detail.

Example 1: the Proliferation of lines activated T-lymphocytes in the presence of monoclonal antibodies against LAG-3

Monoclonal anti-LAG-3 antibodies, which are used, are 17. B4 described BAIXERAS and others (2) and deposited in the National collection of cultures and microorganisms under the number 1-1240 July 10, 1992 , and 11 E3 described HUARD and others (8).

These antibodies belong to the IgGI isotype. These antibodies are experiencing in relation to their biological effects on activated T-lymphocytes stimulated by a specific antigenic peptides or rebuilt antigens presented by CMH molecules of the second class, expressed autologinid antigen presenting cells expressing LAG-3.

Monoclonal antibody against CD48, denoted 10 H3, used as nepodhodyashaya by immunofluorescence assay on PHA-blasts (phytohemagglutinin) and cell lines, transformed by Epstein-Barr (EBV). In experiments on cell proliferation, monoclonal antibodies added in an amount 5 times higher than the saturated concentration.

Used line of T-lymphocytes represent, on the one hand, the clone 154 originating from peripheral blood lymphocytes, raised against a peptide mimicking a fragment of a hemagglutinin (HA) of influenza, which has the amino acid sequence covering amino acids 306 - 329 (peptide p20); on the other hand, clone 28, T-limfotsity clone, derived from peripheral lymphocytes of the same human donor, grown against diphtheria toxoid (DT). Antigen cells (APCS), corresponding to clone 154 are B-lymphocytes transformed by Epstein-Barr of the same donor (DR 3/DR 11) that T 154. Antigen cells corresponding to clone 28, are B-lymphocytes transformed by Epstein-Barr one and the same donor. This clone is limited to the main complex histosol-value human donor 7.

In the case of clone 154, AIC (5106) incubated at 37oC for 1.5 hours with variable doses of peptide p20, then washed and irradiated (10000 rad. ). SUP>5- 10105cells per ml) in the ratio 3: 1. To clone 28 the proportion of responding cells/viable cells is 1.

Antigen cells major histocompatibility complex of man DR 7/ ABV treated with mitomycin or irradiated, then add to T-lymphocytes in the presence of DT (which remains in the culture). The final concentration of cells of clone 28 is 100,000 cells per ml.

3H-Thymidine (1 microcurie/dredging) add in the time intervals varying from day 2 to day 10 of cultivation.

Each experiment was repeated three times.

The results are expressed as the average number of pulses per minute and after subtracting the number of pulses per minute detected in negative control (T-lymphocytes, cocultivation with uncharged, as immunogens, AIC). Experiments on proliferation carry on tablets with 96 cavities. Absorption of tritium-labeled thymidine in individual recesses in 200 µl was measured after addition of 1 microcurie thymidine during the last 18 hours of cultivation. The results are expressed as the average of three experiments. The typical variation is usually below 12% (a little more if smarturi (clone 154/AIC) collect, filtered using membranes with a pore size of 0.22 μm, divided into samples and frozen at -20oC until titration using commercially available kits for immuno-tests: set for titration of IL-2 and INF - company Immunotech; a set of IFN - firm Gensim and recruitment of IL-4 firms, Showmap Chemicals.

Study regarding the determination of the dose of exercise to establish profiles (contours) proliferation of clone 154 in the presence of the specific antigen p20 in volatile concentrations and in the presence or not of a monoclonal anti-LAG-3 antibodies or monoclonal alien (inappropriate) antibodies (negative control).

Individual results of 16 different experiments show that, whatever the concentration of added antigen, the starting point up to the maximum proliferation does not change, however, systematically observe a significant extension of the proliferation of T-lymphocytes, inkubiruemykh with monoclonal anti-LAC2-3-antibodies. The Fab fragments of monoclonal antibodies 17B4 received and used in the experiment proliferation of clone 154. Profile of proliferation of T-lymphocytes activated by antigen fragments are Fab 17B4 (15 μg/ml), similar to that of cells incubated in the presence of a the effects cannot be attributed to nonspecific reactions, induced by the Fc region of monoclonal anti-LAG-3 antibodies.

Similar results were obtained when using monoclonal anti-LAG3 antibodies IIE3.

Clone 28 also stimulate antigen (tetanus toxoid, 10 μg/ml) in the presence of monoclonal antibodies 17B4 after cocultivation with the appropriate antigen presenting cells (APC) in the presence of DT. The results are presented in Fig. 2.

Effects of monoclonal anti-LAG-3 antibodies observed in the case of clone 28, namely prologize proliferation, similar to those observed in the case of clone 154.

Conducted experiments designed to identify various cellular events (les evenements) occurring after antigenic stimulation of the cells of the clone 154, incubated in the presence of monoclonal anti-LAG-3 antibodies.

Cells harvested during the classical antigenic stimulation of clone 154 in the presence of monoclonal anti-LAG-3 or anti-CD48 antibodies or in the presence of antibodies and test for expression of transmembrane receptors LAG-3 and CBB25 and samples of culture supernatant fluids are taken in different time intervals after stimulation and test for the presence of interferon-necrosis factor openonline anti-CD3 antibody and monoclonal anti-CD25 antibody) show the receptors for interleukin-2 weakly, but significantly increased after 5 days after antigenic stimulation. Similar experiments with monoclonal anti-CD3 - and IIE3 (anti-LAG-3)-antibodies show that LAG-3, starting from the day of activation, sverkhekspressiya. In addition, the secretion of interleukin-2, interleukin-4, interferon-and tumor necrosis factor also modulated by incubation with monoclonal anti-LAG-3 antibodies, which suggests that different events in the cells change due to the presence of monoclonal anti-LAG-3 antibodies, and that some events take place 24 hours after stimulation.

These results indirectly suggest that LAG-3 plays a role of the regulator in the case of CD4+. The fact that monoclonal anti-LAG-3 antibodies increase proliferation, therefore act as immunochemical suggests that LAG-3 is involved in the "decontamination" T-limfotsitov CD4+when the negative role of LAG-3 in the ratio-dependent antigen stimulation.

Example 2: Transient expression of fused protein LAG-3

Soluble proteins (proteins) derived from LAG-3, receive according to the method of recombinant DNA using appropriate vectors, vkluchaut transfected Cos cells. This system allows you to produce a few milligrams of recombinant fused proteins. Methods of recombinant DNA carried out as described by MANIATIS and others (22). Changes made in accordance with the manufacturer's recommendations.

The design of LAG-3 D1-D4 Ig and LAG-3 D1 D2 Ig

Fragments of the coding region D1 D2 or D1-D4, amplified (30 cycles) of the fragments of the cDNA (sequence follicular dendritic cells, FDC), including cDNA LAG-3 (TRIEBEL, etc. , /1/), using Tag polymerase devoid of activity of 5'-endonuclease and relatively resistant to ageing at a very high temperature; after amplification followed by denaturation at 98oC (with thermal circular DNA, Perkin Elmer Cetus class").

Using specific primers listed in the table below.

The resulting amplificatoare fragments (739 base pairs and 1312 base pairs for LAG-3 DD and LAG-3 D1-D4, respectively) inserted into plasmid pBS (Stratagene).

The paste obtained after digestion with XhoI and BglII and injected into sites XhoI/BamHI vector pCDM7 - CD8 - IgGI (pCDM7 comes from pCDM8, issued for the sale of the company Stratagene), as illustrated in Fig. 3, in order to exchange posledovatel the Torah contain the sequence encoding DD or D1-D4 connected to the DNA sequences encoding the wiring-CH2-CH3chain human IgHI.

CDM7 is a eukaryotic expressing vector, derived from the vectors described by SEED and others (10) for cloning DNA and its expression in E. coli and eukaryotic cells. CDM7 has the following characteristics: (I) the promoter of the human cytomegalovirus for transient expression in mammalian cells; (II) viral source SV40 for autosomal replication of mammalian cells expressing T-antigen; (III) VX (type Col El) as plasmid source for the increased number of copies; (IV) selection SupF for resistance to ampicillin and tetracycline in E. coli Tetamband Ampamb; (V) the source replication MI3 to release single-stranded RNA (un simple brin); (VI) the promoter of T7 RNA; (VII) polylinker for efficient cloning of heterologous DNA.

Transient expression in Cos cells

Cells Cos (5106) transferout using 30 µg DNA respective expressing vectors (coding or LAG-3 DD Ig, or LAG-3 D1-D4 Ig or CD8 Ig) by electroporation (200 V, 1500 mchard, 30-40 msec) using the apparatus Cellject (Eurogentech, liège, Belgium). Cells SNO is erotci. The supernatant taken after 6 days after transfection.

For the synthesis of the resulting fused protein is monitored by assaying the supernatant liquids, as well as extracts of transfected cells by the method of Western blotting with monoclonal antibodies 17B4. Immunoreactive material detected in the supernatant of cells transfected with DNA coding LAG-3 DD Ig or LAG-3 D1-D4 Ig.

In parallel, recombinant immunoadhesin CD8 (CD8 Ig) is obtained as a negative control using the same expression system, and expressing the vector pCDM7-CD8 (Fig. 3).

Recombinant protein LAG-3 DD Ig, LAG-3 D1-D4 Ig and CD8 Ig purified by classic method on protein a-sepharose. The resulting material is analyzed by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate with subsequent Coomassie - staining or analysis by the method of Western blotting using human anti-Ig antibodies.

Example 3: Obtaining soluble subfragments LAG-3

In order to obtain large quantities of recombinant proteins was developed stable expression system, consisting of a transfected mammalian cell. The cell is digidrofolatreduktazy (dhfr) and therefore, requiring for their growth glycine, purine and thymidine. The main role of dhfr in the synthesis of nucleic acid precursors in combination with the sensitivity of cells dhfr deficiency in respect of the analogs tetrahydrofolate, such as methotrexate (MTX) has two significant advantages. Transfection of these cells expressing vectors containing the dhfr gene, allows you to select dhfr - resistant recombinant clones, and culture of these cells on selective media containing increasing amounts of MTX, comes from amplification of dhfr gene and the DNA that is associated with it.

The design of LAG-3 D1, LAG-3 DD, LAG-3 D1-D4

The DNA fragments coding region D1, DD or D1-D4, amplified using the method of polymerase chain reaction, similar to that described above, using primers shown in table 4.

The resulting amplificatoare fragments digested with SalI and inserted into the SalI site pUC18 (Stratagen).

Amplificatoare sequence identify and paste subcloning in expressing vector pCLH3 AXSV2 DHFR h IVS, as described by COLE and others (Biotechnology, II, 1014-1024, 1993) (Fig. 4).

This vector is a multi-eukaryotically: (I) the murine promoter gene metallothionein-1 sequence and the SV40 polyadenylation (including the site of the donor-acceptor binding to transcription of the target gene; (II) human intron A, which includes the site of the donor-acceptor binding subunit gene-glycoprotein, to achieve high levels of cDNA transcription; (III) the sequence pML containing the pBR322 replication source and the gene of resistance to ampicillin for bacterial amplification; (IV) the unit of transcription of dhfr SV 40 for transcription of sequences used for selection and amplification of transfectants.

Stable expression in CHO-cells

Expressing vectors encoding LAG-3 D1, LAG-3 DD and LAG-3 D1-D4, are used for transfection of cells CHO DUKX and these cells were cultured on selective medium. Cells able to proliferate in these conditions, harvested and cultured on a medium containing increasing amounts of MTX. The level of expression is determined using an analysis by the method of Western blotting using monoclonal antibodies 17B4. Clones producing large quantities of soluble recombinant molecules originating from LAG-3, propagation in bioreactors and originating from LAG-3 material purified by ion-exchange and immunoaffinity chromatography.

Analyses by the method of Western blotting detected in the supernatant fluids of the cells, trafficera the masses Mr 15-18 KD, 34-36 KD (doublets) and 55 KD (2 possible bands). The corresponding Mr these immunoreactive materials were consistent with the expected Mr for glycolytically LAG-3 D1 Ig (139 amino acids and the proposed site glycolythiourea), LAG-3 DD Ig (239 amino acids, including 3 site glycolythiourea) and LAG-3 D1-D4 Ig (412 amino acids, including 4 site glycolythiourea).

Example 4: Specific binding of LAG-3 Ig with cells expressing CMH second class

The reactivity of monoclonal antibodies and LAG-3 D1-D4 Ig studied by indirect (indirect) immunofluorescence assay. Target cells (4x10) cultured for 30 minutes at 4oC in the presence of LAG-3 D1-D4 Ig, CD8 Ig, mouse monoclonal antibody, (949) human anti-CMH second class (DR, DP, DO) conjugated to isothiocyanates (FITC), Coulter clone or murine Ig - isothiocyanate: alien immunoglobulin G conjugated with isothiocyanates. Cells are washed and incubated at 4oC for 30 minutes with either polyclonal goat F(ab') against chelovecheskogo immunoglobulin conjugated to fluorescein or with a polyclonal goat antibody against mouse immunoglobulin conjugated to fluorescein (clone Coulter).

For the showing class. Four lines B-lymphocytes expressing CMH second class (L31, Phil of emulsion explosives, Raji, Sanchez and Personnaz), treated with a monoclonal antibody 949 against class II, or supernatant fluids Cos cells, transfected with DNA encoding either LAG-3 D1-D4 Ig or CD8 Ig. Five cell lines expressing different haplotypes CMH molecules of the second class are LAG-3 Ig in the same way as monoclonal antibodies against class II (positive control), then containing CD8 Ig supernatant (negative control) is not associated with these cell lines, as you would expect. Four lines negative cells CMH second class (CEM, RJ, HSB2, K562) treated with the same reagents as above. No reaction with anti-CMH second class (negative control), and no reaction with LAG-3 D1-D4 Ig shows that communication LAG-3 D1-D4 with CMH molecules of the second class is specific.

Additional experiments are implemented using (I) murine fibroblasts, transfected or retransfusion with genes encoding human DR7 or human DP4, (II) murine cells expressing or inexpressibly CMH molecules of the second class, (III) activated human CD4+

In contrast to CD8 Ig, LAG-3 D1-D4 Ig is associated with any of the cells expressing CMH second class, as effectively as monoclonal antibody 949 against CMH second class. LAG-3 D1-D4 Ig is associated with any of the investigated haplotypes DR and DP, with molecules of human CMH second class, expressed transfitsirovannykh murine cells with molecules of murine CMH second class, as well as with CMH molecules of the second class, expressed by T-lymphocytes CD4+or CD8+.

These results are the first to provide evidence that soluble molecules derived from ligand CMH second class, is able to bind to the cells expressing CMH second class.

Such experiments show that LAG-3 DD associated with cells expressing CMH second class, also specifically and with the same efficiency that LAG-3 D1-D4.

Link activity LAG-3 Ig and distribution in cells ligand LAG-3 Ig

The ability of this immunoadhesin contact with cellular ligands is determined using goat serum, directed against human immunoglobulins labeled with fluorescein.

In these experiments, target cells are first Inc is tereasa 10% fetal calf serum (FCS). Cells are then incubated with goat serum against mouse immunoglobulin labeled with FITC (Coulter), monoclonal mouse antibodies or goat serum against human immunoglobulin labeled with FITC (Tago), for immunoadhesins. Fluorescence was measured after two washes, analyzing 3000 cells on the Elite cytometer (Coultronics, Hialeah, Finland). In Fig. 9 presents the degree of fixing LAG-3 Ig, CD8 Ig, antibodies 949 or antibodies OCST (anti-CD3, ATCC), as the number of counted cells depending on the logarithm of the intensity of the measured fluorescence.

LAG-3 Ig recorded in transfected murine fibroblasts for gene molecule HLA DR4but not recorded on nitrostilbene cells. CD8 Ig is unable to communicate with fibroblasts HLA DR4+in the same conditions. The distribution of cells ligand LAG-3 Ig appreciate the way cell populations by immunofluorescence assay.

LAG-3 Ig detect all positive test cells of the second class, including the back of the line B-cells transformed by Epstein-Barr (derived genetically unrelated donors, including 10 homozygous lines of type DR1-DR10), as well as in activated T-cells and NK-cladocalyx of Ableton DAUDI with the antigens of the second class.

The average fluorescence intensity with LAG-3 Ig similar to that observed in the case of specific antibodies 949 of Utegenov second class. Fixing LAG-3 Ig on DR4(Fig. 9), DR2DR7or DPw4 (not shown), expressed on the surface of mouse fibroblasts, in contrast, weaker than that observed for antibodies 949.

Not detected no binding with negative cell lines for antigens of the second class T-origin (T-cells peripheral blood line CEM, HSB2, REX), B-origin (line Rf 2.2.5), or non-lymphoid origin (human line K562 Citro-microidea origin and lines derived from melanoma cells is /not present/).

In addition, LAG-3 Ig is fixed on xenogenic molecules of the second class CMH, such as antigens, expressed murine lymphoma A 20, and the monkey class II expressed by the blasts stimulated phytohemagglutinin (data not shown).

The specificity of fixing LAG-3 Ig also confirmed by the use of monoclonal antibodies 17B4, the ability to block the interaction between LAG-3 CMH second class in tests on cell adhesion was shown earlier (Fig. 10).

food, or 17B4 (1 mg/ml), or with ACTS (1 mg/ml), prior to the introduction into contact with DAUDI cells.

In Fig. 10 shows that pre-incubation LAG-3 Ig with 17B4 inhibits the fixation of the cells class II+while no inhibition is not observed with the control ACTS.

Example 5: Inhibited the interaction between LAG-3 class II CMH using soluble fragments of LAG-3

Inhibition of the interaction between LAG-3 class II CMH using soluble fragments of LAG-3 can be observed directly at fixing LAG-3 Ig at CMH second class in experiments by competition with soluble fragments.

To confirm whether soluble fragments of LAG-3 DD produced by cells of the ovary hamster, replace the link immunoadhesins originating from LAG-3, perform the following tests:

The DAUDI cells incubated with soluble fragments of LAG-3 DD that could be fixing these molecules on the antigens of the second class CMH, expressed on the surface of DAUDI cells.

In the second stage, cells incubated in the presence of LAG-3 - D1D4Ig in dimeric form or LAG-3 D1D2Ig in Monomeric form.

Fixing these immunoadhesins originating from LAG-3, definition(GAH FITC).

The control group is represented by DAUDI cells, incubated with the dimeric form of LAG-3 D1D2Ig or Monomeric form of LAG-3 D1D2Ig without pre-incubation with soluble fragments of LAG-3 D1D2.

The results are presented in table 5, which States that soluble fragments of LAG-3 D1D2the ability to replace immunoadhesin originating from LAG-3 in Monomeric or dimeric form.

These data confirm that soluble fragments of LAG-3 D1D2fixed on the molecules of the second class CMH.

Inhibition of the interaction between LAG-3/CMH second class and CD4/CMH second class

The formation of rosettes between Cos cells, transfitsirovannykh LAG-3 wild-type and B-lymphocytes transformed by the virus of Epstein-Barr expressing molecules CMH second class, shown BAIXERAS and others (2). This interaction inhibited simultaneously monoclonal antibodies against human haemoglobin cells (MHC).

Described in this publication, a method of modifying, replacing visual observation and counting communicating with B-lymphocyte cells Cos counting residual radioactivity after incubation, labeled with 51

Cells Cos transferout relevant expressing vector (encoding LAG-3 wild-type or CD4). 2 days later Cos cells treated with trypsin and again apply a thin layer (monolayer) of 0.05 to 106cells/indentation on the tablets for culturing tissue with 12 grooves. After 24 hours, labeled with51Cr DAUDI cells (5,5106) incubated in the monolayer of Cos cells (final volume 1 ml) for 1 hour. Wild B-cells are then sucked off and deepening washed 5-7 times, adding there slowly drop by drop 1 ml of medium. The edges of the recesses washed by aspiration using a Pasteur pipette. The remaining cells are lysed using 1 ml of phosphate buffered saline (SFR), with 1% Triton for 15 min at 37oC. the Lysates centrifuged with a speed of 3000 rpm for 10 minutes and get 100 μl of the resulting supernatant.

LAG-3 D1-D4 Ig is used for inhibiting the interaction between LAG-3/CMH class II and CD4/class II CMH in the experience of binding in the case of51Cr. Human CD8 Ig and IgGI experience in parallel and used as a negative IG. 5A). However, the interaction between LAG-3/CMH second class may be partially and nonspecific ingibirovalo using CD8 Ig and human IgGI. On the other hand, LAG-3 Ig is a potential inhibitor of the interaction of CD4/class II (Fig. 5B) in experimental conditions where the interaction of CD4/class II CMH is not modified by CD8 Ig or human IgGI. This confirms the fact that the interaction between LAG-3/class II weaker than the interaction of CD4/class II. These results are the first evidence of the possible competition of soluble molecules in the interaction CMH second class with its ligands.

Example 6: the Immune-suppressive activity of LAG-3 D1-D4

The corresponding experiments are performed using test against proliferation described above for the biological activity of monoclonal antibodies against LAG-3.

After 3 days and 5 days (day 3 and day 5) after antigenic stimulation, LAG-3 D1-D4 Ig shows strong inhibition against proliferation of clone 28, whereas in human CD8 Ig and IgG has no effect (Fig. 6). Similar experiments carried out using clone 154 (Fig. 7) and show partial inhibition in the presence of LAG-3 Ig.

Implemented with motacillidae inhibition of cell proliferation incubated in the presence of LAG-3 D1-D4 Ig, is also observed for clone 28.

These observations suggest that LAG-3 D1-D4 Ig is a potential immuno-suppressing proliferation of T-lymphocytes stimulated by antigen, and suggest that LAG-3 can act as a "sink" secondary immune response induced by helper T-lymphocytes, activated CD4+.

The role of Lag-3 Ig in the negative regulation of immune responses by T-cells

In order to show that a soluble form of LAG-3, imitating the functions of membrane molecules can inhibit the activation of T-clone CD4+stimulated by antigen, conduct the following experiments using clone T154: T-cells were pre-incubated with a rich amount of LAG-3 Ig (100 nmol). Cells are then washed twice with cold RPMI and incubated with 10 μg/ml goat antibody directed against human immunoglobulin (Tago) at 4oC for 30 minutes.

After two new leaching cells again suspended in RPMI containing 10% fetal calf serum, and incubated for 2 hours at 37oC before the addition of the "signal". For cross-stitching monoclonal antibodies using the Thor clone T154 pre-incubated with LAG-3 Ig, related ("cross-linked") with a second reagent (specific polyclonal serum for the constant region of human immunoglobulin). The degree of fixation of LAG-3 Ig on the cells was determined by immunofluorescence assay (Fig. 11A). In Fig. 11B shows that inhibition of more than 50% of the proliferation of clone T154 reach due to LAG-3 Ig. In the same experimental conditions no effect not observed with control CD8 Ig or LAG-3 Ig without "cross-linkage" (Fig. not presented).

In Fig. 11C also shows that no effect is observed when LAG-3 Ig is used for linking (cross-linking) of molecules of the second class CMH, downregulation of antigen presenting B-cells.

Possible effects related ("cross-linked") monoclonal antibodies against the second class at the level of proliferation of T-cells compared with those LAG-3 Ig. Weak inhibition (below 50%) observed in the case of antibodies 949 and D1 antibodies.12 (anti-DR) associated with antimachine goat polyclonal serum (Fig. 12). The inhibition of proliferation, therefore, depends on the epitope, with the most significant effect is reached with a specific epitope LAG-3 connection with class II molecules.

Vozduha clone T CD4+specific clone TDEL peptide 34-53 basic protein of myelin.

The inhibition of proliferation observed (n = 2), when TDEL stimulate antigen (not shown), immobilized ACTS (Fig. 13A), pectin (PHA /phytohemagglutinin/ + PMA /formalparameterlist/) (Fig. 13B) and 5 IU/ml of interleukin-2 (Fig. 13C). No inhibition is not observed with 100 IU/ml of interleukin-2 (Fig. 13D).

In conclusion, the combination of these results indicates that LAG-3 and CMH molecules of the second class, which represent each of these factors, the activation antigens to T-cells, are able to assimilate with molecules-effectors involved in the phase of inactivation response of T-cells. In addition, these results illustrate the importance of interactions between T-cells in the control of immune response cells.

Example 7: Stimulation of cell cytotoxicity due to LAG-3 Ig

The role of LAG-3 Ig against cellular cytotoxicity study using two types of effector cells:

- freshly isolated human peripheral blood lymphocytes (PBL);

cells lines SIB5 (clone human NK-cell /natural killer cells/).

The cytotoxic activity of these cells is risotti or absence in the environment of LAG-3 Ig.

In Fig. 14 shows degree of cytotoxicity SIB5 for the line of human B cells transformed by Epstein-Barr and bearing the antigens of the first and second classes of major histocompatibility complex (line LAZ 388), depending on various reagents added to the cultures.

Measurements are carried out after 4 hours after cocultivation for ratios of effector cells/target cells (SIB5/LAZ 388) 3: 1 (white columns) or 1: 1 (shaded columns).

Negative controls are: one medium (MED), immunoadhesin CD8 Ig and monoclonal antibody 17. B4 (anti-LAG-3).

Positive controls consists of three different monoclonal antibodies:

- antibody L243 directed against antigens class II DR;

- antibody 9.49 directed against antigens class II DR, DP, DO;

- antibody W632 directed against antigens of class I major histocompatibility complex of man.

Antibodies against major histocompatibility complex human class I (W632) or class II (1.243) increase the lysis of target cells (but not control 17B4). Immunoadhesin LAG-3 Ig increases lysis; control CD8 Ig has no effect.

In Fig. 15 presents Edam DAUDI (major histocompatibility complex human class I-for the ratio of the effector/target of 50: 1 (white columns) and 15: 1 (shaded columns). Added on Wednesday reagents are the same as the reagents used in the first experiment, without antibodies 9.49 and antibodies 17. B4. Antibody 10H3 is a specific immunoglobulin isotype IgGI surface antigen CD45. It is used as a negative control.

Any changes are not observed when using antibodies directed against antigens of class I major histocompatibility complex (W632).

These two series of measurements indicate that, compared with negative controls, LAG-3 Ig activates cytotoxicity NK-cells. This effect is similar to that observed with antibodies directed against molecules of the second class CMH.


1. TRIEBEL T. et al. , 1990, J. Exp. Med. 171, 1393-1405.

2. BAIXERAS E. et al. , 1992, J. Exp. Med. 176, 327-337.

3. COSGROVE D. et al. , 1991, Cell 66, 1051-1066.

4. RAHEMTULLA A. et al. , 1991, Nature 353, 180-184.

5. TRAUNECKER, A. et al. , 1988, Nature 331, 84-86.

6. BENEDICT A. A. et al. , 1967, Methods in Immunology 1, 197-306 (1967).

7. YELTON, D. E. et al. , Ann. Rev. of Biochem. 50, 657-680 (1981).

8. HUARD B. et al. , Immunogenetics 39: 213.

9. MANIATIS T. et al. (1982), Molecular cloning: A laboratory manual - Cold S2. COLE S. C. et al. Biotechnology II, 1014-1024, 1993.

1. Soluble polypeptide fraction formed by at least the first of the four extracellular immunoglobulin domains of the type of protein LAG-3 (amino acids 1-149), in which one or more residues of arginine (Arg) at positions 73, 75 and 76 of the sequence SEQ ID no1 has been replaced by glutamic acid (Glu), and possibly one or more of the other three extracellular immunoglobulin domains of the type of protein LAG-3 (amino acids 150-239, 240-330 and 331-412 sequence SEQ ID no1).

2. Soluble polypeptide fraction under item 1, in which at least one of the extracellular domains of the protein LAG-3 is merged with at least one additional peptide sequence in the form of a fused protein.

3. Soluble polypeptide fraction under item 1 or 2, wherein the additional peptide sequence is the sequence of human immunoglobulin.

4. Soluble polypeptide fraction under item 3, wherein the additional peptide sequence is the sequence of human immunoglobulin type IgG4.

5. Soluble polypeptide fraction in one of the paragraphs. 1-4, characterized polypeptides faction on one of the PP. 1-5, characterized in that the cDNA fragments encoding the extracellular domains of the immunoglobulin type of protein LAG-3, and possibly the cDNA fragment encoding the additional peptide sequence inserted into a vector selected from the group comprising CDM7, pCDM7-CD8, pCLH3 AXS V2 DHFR hIVS and pCDM-7-CD8-IgGl, and carry out the expression of cDNA fragments after transfection with the expression system.

7. The method according to p. 6, wherein the expression system comprises ovary cells hamster.

8. Soluble polypeptide fraction, formed the whole or part of at least one of the four extracellular immunoglobulin domains of the type of protein LAG-3 (aminokisloty 1-149, 150-239, 240-330 and 331-412 sequence SEQ ID no1), characterized in that the peptide sequence of LAG-3 further comprises at its C-and/or N-end of the additional peptide sequence type human immunoglobulin, forming a fused protein.

9. Soluble polypeptide fraction under item 8, wherein the human immunoglobulin is an immunoglobulin isotype IgG4.

10. Soluble polypeptide fraction under item 8 or 9, characterized in that it further associated with a toxin or a radioisotope.

12. The method according to p. 11, wherein the expression system comprises ovary cells hamster.

13. Pharmaceutical composition having immuno-suppressive activity, including soluble polypeptide fraction in one of the paragraphs. 1-5 or soluble polypeptide fraction in one of the paragraphs. 8-10 and a pharmaceutically acceptable carrier.

14. Antibodies specifically recognizing soluble polypeptide fraction in one of the paragraphs. 1-5, selected from monoclonal antibodies or fragments of these antibodies, in particular, fragments Fab, Fab', F(ab')2.


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