# Patent

## Title
Phytoestrogen supplement prepared from pomegranate seeds and a herbal

## Abstract

Novel polypeptides having lymphocyte stimulating factor activity are disclosed, as well as the use thereof for treating African sleeping sickness. Also, the corresponding nucleic acid sequences are provided as well as truncated forms which encode polypeptides exhibiting enhanced biological activity.

## Background
The present invention relates to a lymphocyte triggering factor, which has the ability to stimulate CD8+ T cells in a living body. The invention also relates to a nucleotide sequense coding for said lymphocyte triggering factor as well as a monoclonal antibody directed against said factor.

African Trypanosomiasis is a disease widespread in the tropical Africa. It exists in (i) humans as Sleeping Sickness, caused by Trypanosoma brucei gambiense (T.b. gambiense) or T.b. rhodesiense, (ii) in cattle as Nagana caused by T. congolense and T. vivax and (iii) in camels as Surra and caused by T. evansi. Mortality is caused either by massive parasitosis or secondary infections due to immunosuppression, which is an important trait of the disease (Werry et al., 1982, Askonas,B. A. "Interference in general immune function by parasite infections; African trypanosomiasis as a model system", Parasitology 1984; 88:633-638). Existing therapies are either cumbersome or toxic and there is no available vaccine. Furthermore, methods for diagnosing the disease are poorly developed.

A new principle for how a microorganism may subvert the host defense has been detected using an experimental model for African tryptanosomiasis where rodents have been infected with T.b. brucei. The studies focused on CD8.sup.+ T cells and interferon gamma (IFN-.gamma.) as putative important host derived elements in the interactions with the parasite during infection. IFN-.gamma. production in the spleen increased markedly early after infection (Bakhiet,M., Olsson, T., Van der Meide, P., and Kristensson, K., "Depletion of CD8.sup.+  T cells suppresses growth of T. brucei brucei and IFN-.gamma. production in infected rats", Clin Exp Immunol 1990; 81: 195-199). Both in vivo monoclonal antibody CD8.sup.+ T cell depleted rats, as well as genomically CD8 deleted mice, showed absence of IFN-.gamma. induction, dramatically decreased parasitaemia and prolonged survival (Bakhiet et al., 1990, supra, Olsson, T., Bakhiet, M., Hojeberg, B., Ljungdahl, .ANG.., Edlund, C., Andersson, G., Ekre, H-P, Fung Leung, W-P, Mak, T., Wigzell, H., Fiszer, U., Kristensson, K., "CD8 is critically involved in lymphocyte activation by a Trypanosoma brucei brucei released molecule", Cell 1993; 72:715-727). Also, intraperitoneal injeciton of anti IFN-.gamma. antibody suppressed parasite growth and increased survival of the animals (Olsson et al., 1993, supra). In vitro studies showed that the trypanosome released a lymphocyte triggering factor (TLTF), which through binding to CD8 on lymphoid cells, triggers these cells to secrete IFN-.gamma., which in turn constitutes a growth stimulus for the parasite (Olsson, T., Bakhiet, M., Edlund, C., Hojeberg, B., Van der Meide, P., and Kristensson, K.,"Bidirectional activating signals between Trypanosoma brucei and CD8.sup.+  T cells: A trypanosome-released factor triggers interferon gamma production that stimulate parasite growth", Eur. J. Immunol. 1991; 21:2447-2454; Olsson et al, 1993, supra). A mouse monoclonal antibody (MO1) was raised and used to purify TLTF with affinity chromatography. In addition, passive immunotherapy in vivo with MO1 strongly reduced parasite levels and prolonged survival of the animals (Bakhiet, M., Olsson, T., Edlund, C., Hojeberg, B., Holmberg, K., Lorentzon, J., and Kristensson, K., "A trypanosoma brucei brucei derived factor that triggers CD8.sup.+ lymphocytes to interferon gamma secretion: Purification, characterization and protective effects in vivo by treatment with a monoclonal antibody against the factor", Scand. J. Immunol. 1993; 37:165-178).

Hitherto, it has not been possible to clone TLTF for the production of recombinant material or peptide parts of the molecule, which could be tested in future vaccination attempts. Had this been possible, the molecule might furthermore be used in the targeting of CD8.sup.+  cells both in vivo and in vitro.

The present inventors have succeeded in the cloning of TLTF. Thus, this invention relates to a lymphocyte stimulating factor comprising a protein component having the ability to stimulate, in a living animal body, CD8+ T-cells resulting in the release of interferon-.gamma. (IFN-.gamma.) to elicit immunosuppression or immunostimulation in said body.

In addition, the invention relates to a nucleotide sequence coding for said lymphocyte stimulating factor as well as a monoclonal antibody directed against it.

For a better understanding therof, the present invention will be disclosed under reference to figures. While a more detailed disclosure thereof will be found below, a summary of the figures is as follows:

FIG. 1A depicts SEQ. ID. NO. 1, which is the complete cDNA sequence for TLTF.

FIG. 1B depicts (SEQ. ID. NOS. 2,4,6,8,10,12,14) which is the amino acid translation of the cloned cDNA.

FIG. 1C depicts (SEQ. ID. NOS. 3,5,7,9,11,13,15) which is the protein according to FIG. 1B.

FIG. 1D depicts (SEQ. ID. NO: 16) which is the complete predicted amino acid sequence of TLTF.

FIGS. 1E-H depicts (SEQ. ID. NOS: 17-20) which are the predicted amino acid sequences of four truncations of TLTF according to the invention.

FIG. 2 shows the number of IFN-.gamma. secreting cells determined in an immunospot assay by a graph, wherein the number of IFN-.gamma. spots/10.sup.6 rat MNC are plotted for TLTF, rTLTF and OV7.

FIG. 3 shows monoclonal anti-TLTF antibodies effect on rTLTF triggering of human peripheral blood (PBL).

FIG. 4 shows the effects of TLTF and rTLTF on MNC from mice with genomic deletion of CD8.sup.+  or CD4.sup.+  T cells.

FIG. 5 shows the anti-rTLTF immune sera inhibitory effect on native TLTF-induced IFN-.gamma. as measured by the immunospot assay.

The object of the present invention is more specifically a lymphocyte stimulating factor comprising a protein component having the ability to stimulate, in a living animal body, CD8+ T-cells resulting in the release of interferon-.gamma. (IFN-.gamma.) to elicit immunosuppression or immunostimulation in said body, wherein said protein component is selected from amino acid sequences within the sequence of amino acids numbered 1 to about 150 in FIG. 1B, which are in possession of said ability. The amino acid sequence showed no homologies to previously described proteins, and since it represents a non-variable protein it is a potential candidate for immune therapy.

One object of the invention is a lymphocyte stimulating factor, which comprises a protein component consisting of at least about 70 amino acid residues according to FIG. 1B.

Another object of the invention is a lymphocyte stimulating factor, which comprises a protein component consisting of at least about 100 amino acid residues according to FIG. 1B.

Yet another object of the invention is a lymphocyte stimulating factor, which comprises a protein component consisting of at least about 120 amino acid residues according to FIG. 1B.

Further, another object of the present invention is a lymphocyte stimulating factor as defined above, comprising a protein component having the ability to stimulate, in a living animal body, CD8+ T-cells resulting in the release of interferon-.gamma. (IFN-.gamma.) to elicit immunosuppression or immunostimulation in said body, wherein said protein component is selected from amino acid sequences within the sequence of amino acids numbered 1 to about 300 in FIG. 1B.

A further object of the present invention is a lymphocyte stimulating factor as defined above for use as a vaccine against African sleeping sickness.

Another object of the present invention is such a lymphocyte stimulating as is defined above for use in the preparation of an antiserum useful for combatting African sleeping sickness.

Yet another object of the present invention is a nucleotide sequence coding for any of the lymphocyte stimulating factors according to the invention.

Another object of the invention is such a nucleotide sequence, as is shown in FIG. 1A.

Another object of the invention is a monoclonal antibody directed against any one of the lymphocyte stimulating factors according to the invention.

One further object of the present invention is a method of treating African sleeping sickness, comprising the administration to a mammal, including man, in need of such treatment of an effective amount of an antibody against any of the lymphocyte stimulating factors according to the invention, said antibody interacting with said factor released by the parasite causing said sickness.

A further object of the invention is a method of vaccinating a mammal, including man, against African sleeping sickness, comprising the administration to the mammal of an immunologically active amount of any one of the described lymphocyte stimulating factors.

Parasites

The following trypanosome preparations were used in the present work: (a) T.b. brucei An Tat 1/1, which was used to generate the native TLTF. The parasites were obtained from Dr. Nestor Van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine, Prins Leopold, Antwerp, Belgium. The parasite were passaged once in rats and purified from infected blood (according to Lanham, S. M., Godfrey, D. G., "Isolation of salivarian trypanosomes from man and other animals using DEAE-cellulose", Exp. Parasitol. 1970; 28:521-534) and then disrupted by freezing and thawing before experimentation. (b) T.b. rhodesiense clone MVAT4 Rx (Alarcon, C. M., Son, H. J., Hall, T., and Donelson, J. E., "A monocistronic transcript for a trypanosoma variant surface glycoprotein", Mol. Cell Biol. 1994;14:5579-5591), from which total RNA was isolated to construct the cDNA library.

Animals

Rats and mice were used in this study. SpragueDawley rats were purchased from Alab (Stockholm, Sweden). DBA/2 MHC H-2d/d mice were locally bred at the Department of Immunology, Karolinska Institute, Stockholm. Mutant mice (CD8.sup.-)with disrupted lyt 2 gene lacking CD8 expression (Fung-Leung, W-P, Schillham, M. W., Rahemtulla, A., Kundig, T. M., Vollenweider, M. J., van Ewijk, W., and Mak, T. W. "CD8 is needed for the development of cytotoxic T cells but not helper T cells", Cell 1991; 443-449), and mutant mouse strain lacking CD4 expression (Rahemtulla A., Fung-Leung W. P., Schillman M. W., Kundig T. M., Sambara S. R., Narendran A., Arabian, A., Wakeham., A., Paige., C. J., Zinkernagel, R. M., Miller, R. G., and Mak, T. W., "Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4", Nature 1991; 353:180-184) originally from Department of Medical Biophysics and Immunology, University of Toronto, Canada, were bred and kept at the Department of Immunology, Karolinska Institute, Stockholm, Sweden).

Antibodies

In in vitro antibody TLTF modulation experiments (see below) the following Mabs were used: (a) IB-4; a Mab directed against TNP (anti-trinitrophenylphosphate) was used as an isotype matched control antibody (mouse IgG2b). The hybridoma producing IB-4 was obtained from Dr. Birgitta Heyman (Uppsala, Sweden), and antibodies prepared from culture supernatants (b) OX8; an anti-rat CD8 antibody (mouse IgG1). The hybridoma producing OX8 was originally obtained from Dr. Alan Williams (Oxford, UK). In some experiments Fab fragments of OX8 was used. The fragments were prepared by papain digestion followed by removal of Fc fragments and any undigested OX8 on protein A (c) MO1, MO2 and MO3; mouse Mabs directed against TLTF. MO1 was prepared as described previously (Bakhiet et al 1993, supra), while MO2 and MO3 were produced recently. Two male DBA/2 mice were immunized in the foot pads with a gel fraction of T.b. brucei that showed a high ability to induce MNC to IFN-.gamma. production (Olsson et al 1991, supra; Bakhiet et al 1993, supra). The immunogen and Freund's complete adjuvant (Sigma, St. Louis) was mixed 1:1 and 50 .mu.l of this mixture was injected into each hind foot pad. Eleven days later regional lymph nodes were removed. Suspended lymphocytes from these nodes were hybridized with mouse Sp 2/0 cells as previously described (Holmdahl, R., Olsson, T., Moran, T., Klareskog, L., "In vivo treatment of rats with monoclonal anti-T-cell antibodies. Immunohistochemical and functional analysis in normal rats and experimental allergic neuritis", Scand. J. Immunol. 1985; 22:157-169). Subcloning was done once and hybridomas, whose supernatants inhibited or stimulated TLTF induced IFN-.gamma. production by rat mononuclear cells cultures were selected. Ten hybridomas supernatants significantly inhibited TLTF induced IFN-.gamma. production, while 7 hybridomas exhibited stimulatory effects. One of the inhibitory hybridomas (MO2) and one of the stimulatory hybridomas (MO3) were further propagated and characterized. Both of these hybridomas produced antibodies of the IgG 2b subclass, and were purified on Protein A Sepharose according to standard techniques.

The affinity purified TLTF (Bakhiet et al., 1993, supra) was also used to immunize rabbits to generate immune sera according to standard protocols. The anti-TLTF polyclonal antibody (NR4) from rabbit number 300 was used for immunoblotting and for screening of the trypanosome cDNA library.

Preparation of Mononuclear Cell Suspensions

Mononuclear cells were prepared from rats and mice spleens, and from human peripheral blood. To prepare spleen MNC, animals were sacrificed, the spleens dissected and crushed through a stainless steel meshwork. The cells were washed once in tissue culture medium. The medium consisted of Isocove's modified Dulbecco's medium (Flow lab. Irvine, UK) supplemented with 5% foetal calf serum (GIBCO, Baisley, UK), 1% minimal essential medium (Flow Lab), 2 mM glutamine (Flow Lab), 50 .mu.g/ml penicillin, and 60 .mu.l,/ml streptomycin. Erythrocytes in the cell pellets were haemolysed by adding 2 ml cold sterile water for 30 sec followed by addition of 1 ml 2.7% saline. The cells were then washed in medium twice and rediluted to obtain a cell concentration of 5.times.10.sup.6 /ml. The cells were then washed twice in medium and rediluted to obtain appropriate cell concentrations (see below). Human peripheral blood cells from healthy donors were obtained by density gradient centrifugation. In some experiments human CD8 and CD4 enriched populations were used. They were isolated by negative selection using Mab coated magnetic beads as described previously (Olsson et al 1993, supra).

Single Cell Assay for IFN-.gamma. Secretion.

The method described by Czerkinsky et al 1988 as adapted to rat IFN-.gamma. (Mustafa, M. I., Diener, P., Hojeberg, B., Van der Meide, P., and Olsson, T., "T cell immunity and interferon-.gamma. secretion during experimental allergic encephalomyelitis in Lewis rats", J. Neuroimmunol. 1991; 31: 165-177) was used to detect IFN-.gamma. production by single secretory cells. In principle, nitro-cellulose-bottomed, 96 well microtitre plates (Millipore, Bedford, Mass.) were coated overnight with 15 .mu.g/ml (100 .mu.l aliquots) of the mouse monoclonal antirat IFN-.gamma. antibody DB1, which crossreacts with mouse IFN-.gamma. (Van Der Meide, P. H., Dubbeld, M., Vijverberg, K., Kos, T., Schellekens, H, "The purification and characterization of rat gamma interferon by use of two monoclonal antibodies", J. Gen. Virol. 1986; 67: 1059-1071). DB1 was a generous gift from Dr. Peter Van der Meide, TNO Center, Netherlands. After repeated washings with PBS, 2% bovine serum albumin was applied for 2-4 h, the plates were washed in PBS and aliquots of 5.times.10.sup.5 MNC (200 .mu.l) were applied in triplicate. Ten .mu.l aliquots of native TLTF, recombinant material (see below) or Con A as a control were added to different cultures. In some experiments other reagents were added (see below). This was followed by incubation overnight at 37.degree. C. in humidified atmosphere of 7% CO.sub.2. Cells were then removed by flicking the plate, followed by repeated washings in PBS. Polyclonal rabbit anti-rat IFN-.gamma. (Van der Meide et al 1986, supra), diluted 1/1000, was applied for 4 h. After washing, biotinylated goat anti-rabbit IgG (Vector Lab, Burlingame, Calif.) was applied for 4 h followed by avidine-biotin-peroxidase complex (ABC Vectastain Elite Kit, Vector Lab). Peroxidase staining with 3-amino-9 ethylcarbazole and H.sub.2 O.sub.2 was performed (Kaplow, L. S: "Substitute of benzidine in myeloperoxidase stains", Am. J. Clin. Pathol. 1974; 63:451). Spots corresponding to cells that had secreted IFN-.gamma. were counted in a dissection microscope.

Lymphocyte Proliferation Assay

Aliquots of 5.times.10.sup.5 MNC (200 .mu.l) were added in triplicate to 96-round-bottomed well microtitre plates (Nunc, Copenhagen, Denmark). The cells were incubated 72 h after addition of 10 .mu.l aliquots of native TLTF, recombinant material (see below) or Con A as a control. Ten h before harvest 10 .mu.l aliquots containing 1 .mu.Ci of .sup.3 H!-methylthymidine (specific activity 42 Ci/mmol) (Amersham, Little Chalfont, UK) in saline were added to each well. Cells were harvested onto glass fibre filters with a multiple channel semiautomated harvesting device (Titertek, Skatron AS, Lierbyen, Norway) and thymidine incorporation was measured as counts per minute (CPM) in a liquid betascintillation counter (Mark II, Searle, Analytic, Des Plaines, Ill., USA).

Cell ELISA for TLTF-CD8 Interaction

Hundred .mu.l aliquots of 10.sup.5 cells in suspension were added and allowed to dry into wells of polystyrene microtitre plates (Nunc, Copenhagen, Denmark). The plates were washed with PBS, pH 7.4, and wells subjected to 0.5% bovine serum albumin (BSA, Sigma) for 24 h at room temperature. After washing with PBS, 100 .mu.l aliquot of different dilution or no TLTF were added for 2 h. After washing with PBS, biotinylated MO3 was added, followed by ABC al P. Enzyme substrate solution was added and absorvence measured at 405 nm in a multiscan photometer (Labsystem, Helsinki, Finland).

Antibody Modulation Experiments

Tel .mu.l aliquots of the IB-4, OX8, MO1, MO2 and MO3 Mabs to obtain final concentration in medium of 5 .mu.g/ml were added to microtitre plate wells immediately after plating the MNC. Ten .mu.l aliquots of native TLTF, recombinant material or as control similar volumes of Con A (5 .mu.g/ml as final concentration in medium; Pharmacia, Uppsala, Sweden) were added. After 24 h of culture the effects on IFN-.gamma. secretion and after 72 h the effect on cell proliferation were determined.

Procedure used for Cloning the Gene Encoding TLTF

The gene was cloned from a .lambda.ZAPII (Strategene) cDNA library constructed with total RNA isolated from blood stream forms of Trypanosoma brucei rhodesiense clone MVAT4 Rx. Host strain cells (bacterial strain) for the Lambda ZAP II Vector (phage) used here was XL1-Blue MRF'. Predigested .lambda.Zap II/EcoR I/CIAP cloning kit (Stratagene) was used to construct and amplify the Lambda Zap II library according to the instruction manual.

Preparation of Plating Bacteria for Phage Lambda:

Using sterile technique, bacterial host from a single colony was inoculated into NCZYM medium, pH 7.5 (50 ml in 250 ml flask). Maltose in final concentration of 0.2% (0.5 ml of 20% stock ) was added to induce malt operon, which contains the gene lamb for the lambda receptor. The bacteria were allowed to grow overnight with shaking at 30.degree. C. This lower temperature ensures that the cells will not overgrow. After incubation, the cell suspension was poured into centrifuge tubes and spinned in bench Sorvsall for 10 minutes at about 3500 rpm. The supernatant was discarded and the pellet was re-suspended in 20 ml 10 mM MgSO.sub.4. Optical density was measured at 600 nm against a blank of 10 mM MgSO.sub.4 and adjusted to 2 by adding more MgSO.sub.4 or re-pelleting cells and re-suspending them in a smaller volume. The cells were stored in aliquots at 4.degree. C. until use.

Plating Phage:

LB Broth medium supplemented with 1% agar that has been autoclaved was poured into plates placed on flat surface. The surface of the poured plate was flamed to remove any air bubbles. When agar had set, plates were put to dry in 37.degree. C. oven overnight in inverted position. Next day, 80 .mu.l of plating bacteria was mixed by inversion with 180 .mu.l phage suspension from different dilutions and incubated at 37.degree. C. for 15-20 minutes. Meanwhile, top agar was melted and dispensed in 8 ml volumes into sterile Falcon tubes, which are then placed in a water bath at 46.degree. C. to equilibrate. Following phage/bacterial incubation, molten top agar was removed from water bath and mixed gently with the diluted phage and host strain using vortex at minimum speed to prevent introducing bubbles. The mixer poured quickly into center of plate in smooth motion and plate was rocked to create an even layer. Plates were left to set for 15 minutes and then incubated overnight at 37.degree. C. in inverted position. After incubation, the number of plaques was counted and the plaque forming units (pfu) was determined per ml concentration of the library based on the dilution.

Immunoscreening:

After titration of the bacteriophage, 2,7.times.10.sup.4 pfu were incubated with XL-1 Blue cells to obtain plaques. The plaques were then lifted in doublicate on nitrocellulose filters (Schleicher & Schuell) impregnated with 0.01M IPTG. Filters were blocked in 5% milk at 4.degree. C. overnight to decrease non-specific binding. Immunoscreening was done using as primary antibody the rabbit polyclonal sera, from rabbit number 300, (NR4) directed against the MO1 affinity purified TLTF. The primary antibody was diluted 1:2000, pre-absorbed with E.coli lysate (Bio-Rad) and incubated with the filters for 2 hours at room temperature. Horseradish peroxidase-linked anti-rabbit donkey IgG (Amersham) diluted 1:5000 was applied for 2 hours at room temperature. Positive plaques were detected using ECL chemiluminescent system (Amersham) where 6 potential phages were picked. The phage titer for the six selected phage was amplified and a secondary immunoscreening was done as described above using 100 .mu.l of 10.sup.-3 dilution of each phage elute. After detection by ECL system, phage 1 and 2 were positive; phage 3 was negative and phage 4,5 and 6 were borderline. Three phages from each group were picked and eluted in SM buffer and phage titer from each pick was amplified.

Rescue of Phagemids and Expression of the Protein:

The inserts in each phage were rescued into SOLR cells using the ExAssist.TM. helper phage (M13) obtained from Stratagene. One clone from each rescue was analyzed further by Western blot. Only one clone (#2.3) had reactive protein, therefore, decided to sequence as TLTF. The nucleotide sequence of #2.3 was determined by using the dedeoxy sequencing method (sequence 2.0 kit, USB). Synthetic oligo primers were used to obtain overlapping sequence. Re-cloning of the gene and different truncated forms was done in the ThioFusion Expression system. Proteins were then purified and examined for TLTF biological activity.

Mann-Whitney's test was used for statistical significance.

cDNA Sequence of the Recombinant TLTF:

The complete cDNA sequence of the recombinant TLTF, clone #2, is shown in FIG. 1A. From 3 reading frames, the 2nd, which is shown, is the longest one and it is open until near the polyA at the 3'end. Supposing that the first MET in this reading frame is a start MET, the deduced protein has 467 amino acids. A geneBank search did not reveal any significant homologies. Using PCR-based approach, 3 truncated forms of the molecule were obtained and over-expressed. Truncated forms of the recombinant protein, disclosed in FIGS. 1A-H, are examined to define the minimum length of the TLTF with retained bioactivity. A 145 amino acids long part of the molecule (#4, which is the shortest part; amino acid 1-145) is still biologically active (FIG. 4B). The translation of the TLTF clone #2 is shown in FIG. 1B. The C terminus of this deduced protein is very hydrophobic, suggesting that the protein is either in a membrane or is secreted. In Western blot analysis of the recombinant E.coli extract, using NR4 (TLTF polyvalent antisera), the corresponding fusion protein has the same sized bands as the native TLTF and T.b. rhodesiense lysate (not shown).

IFN-.gamma. Production by Rat Splenocytes in Response to rTLTF and MO1 Blocking Effect

In the present work we have evaluated IFN-.gamma. production in form of number of cells secreting the cytokine, detected in an antibody capture immunospot assay (FIG. 2). Lymphoid rat MNC cultures were exposed to antibody affinity purified TLTF diluted 10.sup.-3, 1 .mu.g/ml rTLTF and 1 .mu.g/ml of an irrelevant oncoserca protein (OV7) that has been cloned and expressed in a similar way to rTLTF. Other cultures were left non-stimulated. Native TLTF and rTLTF induced significant number of cells to produce IFN-.gamma. compared t non-stimulated cells while OV7 did show a significant effect on IFN-.gamma. produced by rat MNC. The monoclonal mouse anti-TLTF (MO1) was incubated at a concentration of 5 .mu.g/ml with MNC in a set of experiments. This was followed by addition of the 3 proteins to stimulate the cells. MO1 significantly inhibited native TLTF and rTLTF induced IFN-.gamma. production by MNC.

Thus, in FIG. 2, the number of IFN-.gamma. secreting cells determined in the immunospot assay are shown. Normal rat splenocytes suspension was plated at a concentration of 10.sup.6 MNC per well and cultured for 24 h. Triplicate cultures were exposed to optimal dilutions of native TLTF, rTLTF at a concentration of 1 .mu.g per ml or OV7 (oncosera protein that has been cloned and expressed in a similar way to rTLTF) at a concentration of 1 .mu.g per ml. Control cultures received no stimulation. Similar data were obtained on three different occasions. Note the inhibitory effect by anti-TLTF mouse monoclonal antibody (MO1) at a concentration of 5 .mu.g/ml on both native TLTF and rTLTF when incubated with MNC prior to stimulation. As shown previously (Bakhiet et al., 1993, supra), MO1 did not show any effect when incubated with Con A or with non-stimulated MNC. Bars denote S.D.

Effect of Anti-TLTF Antibodies on RTLTF-induced IFN-.gamma. Production by Human Peripheral Blood Lymphocytes (PBL)

The immunospot assay was also used here to evaluate the action of the recombinant TLTF on human PBL and to study the effects of anti-TLTF monoclonal antibodies in the rTLTF triggering activity. Thus, native TLTF at 10.sup.-3 dilution, lug and 0.1 .mu.g rTLTF per ml all induced PBL to produce IFN-.gamma.. Both MO1 and MO2 are monoclonal antibodies that showed inhibitory effect on native TLTF-induced IFN-.gamma. production. The 2 monoclonals herein significantly inhibited the triggering effect of rTLTF at a concentration of 0.1 .mu.g/ml on PBL. Conversely, MO3, a third monoclonal antibody against native TLTF with stimulatory effect, has also exhibited here an enhanced effect on rTLTF-induced IFN-.gamma. production (FIG. 3).

Thus, FIG. 3 shows the monoclonal anti-TLTF anti-bodies effects on rTLTF triggering of human peripheral blood (PBL). After 24 h of culture the number of IFN-.gamma. secreting cells was measured by the immunospot assay. Immediately before addition of rTLTF, different cultures received mouse monoclonal antibodies (MO1, MO2 or MO3) to reach a final concentration in the medium of 5 .mu.g/ml. Other cultures received no antibodies. Note that rTLTF induced IFN-.gamma. production by human (PBL) was inhibited by MO1 and MO2, while MO3 enhanced the effect of the rTLTF. Irrelevant antibodies (OX19, W3/13, W3/25, Hy2-15) were previously shown to have no effects (Olsson et al, 1993, supra). Each staple represent data from triplicate or quadruplicate cultures. Bars denote S.D. The experiment was repeated twice with similar results.

Studies on Cells of Mice with CD8-, CD4-genotype and Normal DBA/2

MNC from mutant mice lacking CD8 or CD4 and normal DBA/2 used to study (a) IFN-.gamma. production (b) T cell proliferation and (c) TLTF-CD8 binding. In vitro stimulation of spleen MNC obtained from the 3 mouse strains showed no increased IFN-.gamma. production using native or recombinant TLTF in CD8.sup.-  mice, while Con-A induced high production of IFN-.gamma. studied in these mice (FIG. 4A). Similar effect was recorded when proliferation assay was used. Thus, native and recombinant TLTF did not trigger MNC from CD8.sup.-  mice, while Con A showed a high proliferative response in all groups. OV7 was used as a control in this assay and did not exhibit any effect (FIG. 4B). The direct binding of TLTF to CD8 was studied by applying native or rTLTF to mouse CD8.sup.+ CD4.sup.-  or CD4.sup.+ CD8.sup.-  mononuclear cell suspensions using cell ELISA. TLTF was allowed to interact with thymocytes from mice with genomic deletions of CD4 or CD8. Thus, binding of TLTF (native and recombinant) was recorded in microtitre plate wells that had been coated with thymocytes containing CD8.sup.+  cells from the genomically CD4 detected mice, but not in wells that had been coated with thymocytes containing CD4.sup.+  cells from genomically CD8 deleted mice. A signal above background was obtained with dilution of TLTF down to 10.sup.-3 during exposure to the CD8.sup.+  containing wells (FIG. 4).

Thus, in FIG. 4, the effects of TLTF and rTLTF on MNC from mice with genomic deletion of CD8.sup.+  or CD4.sup.+  T cells are shown. IFN-.gamma. production (number of IFN-.gamma. secreting cells) was measured after 24 h of culture (A), while proliferation as assessed by .sup.3 H-thymidine uptake was measured after 72 h. MNC were either stimulated with the whole rTLTF or the shortest truncated form #4 (B). Each staple represent data from triplicate or quadruplicate cultures. Bars denote S.D. The experiment was repeated several times with similar results. (C) Absorbance readings in a cell ELISA to study the TLTF-CD8 binding. Mouse thymocytes from genomically CD4.sup.-  mice (containing CD8.sup.+  cells) or CD8.sup.-  mice (containing CD4.sup.+  cells) were dried onto plastic of microtitre plate wells. TLTF, rTLTF or OV7 were applied and binding detected by a biotinylated mouse monoclonal anti-TLTF (MO3). Values denote readings from 6-12 wells after subtraction of the background. Bars denote S.D. Note selective binding of TLTF and rTLTF, but not OV7, to wells containing CD8.sup.+  cells.

rTLTF Immune Sera Blocking Studies on Native TLTF-induced IFN-.gamma. Production

Immune sera from 2 immunised mice with the rTLTF and their preimmune sera were used in blocking experiments to inhibit native TLTF-induced IFN-.gamma. production. The preimmune sera from the 2 mice did not show any effect, while the 2 immune sera significantly inhibited the induction of IFN-.gamma. by TLTF (FIG. 5).

Thus, FIG. 5 depicts anti-rTLTF immune sera inhibitory effect on native TLTF-induced IFN-.gamma. as measured by the immuneospot assay. Preimmune sera gave no effect. Each staple denote reading from 8 cultures. Bars denote S.D.

Native TLTF is a glycoprotein that was characterized for its main biological activities, which is determined by the ability to bind CD8 molecule and stimulate CD8.sup.+  cells to produce IFN-.gamma. and to proliferate. Evidence for such a role was previously obtained by studying T.b. brucei infection in rats or mice. In the former, CD8.sup.+  T cells had been depleted in vivo by anti-CD8 antibodies (Bakhiet et al., 1990, supra) or in vitro (Olsson et al., 1991, supra). While in the later, CD8 was genomically deleted (Olsson et al., 1993, supra). Herein, the functional assays used to demonstrate that the cloned material has similar biological activities as the native TLTF are consequently (a) immunospot method for detection of IFN-.gamma. secreting cells by MNC from rats, humans and mice with genomically deleted CD8 or CD4, (b) T cell proliferation technique and (c) CD8 binding assay. T.b. brucei triggers a high number of lymphoid MNC to IFN-.gamma. secretion in vitro, while lymphoid cell proliferation as measured by .sup.3 H-thymidine uptake is relatively minor (Olsson et al., 1991, supra). However, CD8.sup.+ CD4.sup.-  MNC showed a more potent proliferative response after triggering with TLTF than normal rat or mouse MNC (data not shown). It is tempting to suggest that this simply is due to a higher proportion of CD8.sup.+  TLTF responder cells. All above methods showed that rTLTF selectively acts on CD8. CD8 is a heterodimer with 52% amino acid sequence homology between rat and human (Johnson, P., Gagnon, J., Barclay, A. N., and Williams, A. F. (1985), "Purification, chain separation and esquence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes", EMBO J., 4, 2539-2545). It was suggested that native TLTF may act on interspecies conserved parts of CD8 since it has triggered rat, mouse and human CD8.sup.+  T cells (Olsson et al., 1993, supra). As native TLTF, rTLTF exhibited same effects on CD8.sup.+  cells from all those species. This activity is nevertheless either blocked by the anti-TLTF monoclonal antibodies MO1 and MO2 or enhanced by MO3. Also, immune sera against recombinant TLTF inhibited the native TLTF activity. Previously, we estmiated that roughly one out of 10-20 CD8.sup.+  T cells were triggered by native TLTF (Olsson et al., 1993, supra). Furthermore, native TLTF is active at very low dilution where no measurable protein could be detected. However, rTLTF was active to a dilution of 0.1 .mu.g per ml. This difference in the concentration of the protein required for biological activity may be related to the absence of carbohydrate molecules in the recombinant protein since it is expressed in E.coli. Carbohydrate was shown to be of importance for dynamic biological activity (Bakhiet et al., 1993, supra). Moreover, the recombinant material is insoluble in water, which might also be related to the absence of the carbohydrate. Thereby, methods for purification by elution from gel may affect its activity. In order to overcome this problem expression in other systems, such as mammalian cells, should be tried.

One key approach for protection from African trypanosomiasis is to target invariable molecules that are interacting with the host defens system. Recently, Ziegelbauer K and Overath P., "Identification of invariant surface flycoproteins in the bloodstream stage of Trypanosoma brucei, J. Biol. Chem. 1992; 267:10791-10796) described a class of invariable surface molecules, two of them were isolated and the corresponding genes cloned (Ziegelbauer K, Multhaup G and Overath P., "Molecular characterization of the two invariant surface glycoproteins specific for the bloodstream stage of Trypanosoma brucei., J. Biol. Chem. 1992; 267:10797-10803). They are inserted in the membrane between the VSG molecules and covered by them. Therefore, they are not accessible to antibodies. In passive immunotherapy, injection of the anti-TLTF monoclonal antibody (MO1) into T.b. brucei infected animals resulted in lower parasitemia and prolonged survival. This was of strong indication for cloning the TLTF to be used in an active immunotherapy. This work gives evidence for a correct clone for the native TLTF. The recombinant TLTF will hence be used for vaccination attempts and also will be explored in a broader sense in Biomedicine such as in CD8 targeted immune interventions.

Trypanosome-derived lymphocyte triggering factor (TLTF) is a glycoprotein component secreted by Trypanosoma brucei (T.b.) and binds to CD8 molecule. TLTF-CD8 binding activates CD8.sup.+  T cells to produce IFN-.gamma. and to proliferate. IFN-.gamma. in turn stimulates parasite growth. In this study, the gene coding for the TLTF has been isolated. The nucleotide sequence indicates no relationship to other genes. The deduced protein has 467 amino acids with a very hydrophobic T-terminus, suggesting that the protein is either in a membrane or is secreted. Shorter truncated forms of the recombinant protein (rTLTF) were obtained and examined for biological activities that are characteristic for native TLTF. Thus, rTLTF and its truncated forms were evaluated for their ability to trigger MNC from rats, humans and mice--with genomically deleted CD8 or CD4--to produce IFN-.gamma. and to proliferate. CD8 binding studies revealed that rTLTF selectively binds to the CD8.sup.+  cells and trigger these cells to IFN-.gamma. production and proliferation. The data presented here strongly indicate that the cloned gene is coding for the native TLTF and thereby can be used in further immunobiological studies and in CD8 targeted immune interventions. SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 20 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1750 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TATATTGACTGCATCGTGGCGTACCCCGTAGGCTCTTCTCGTTTTTGAATGTCACCACGG60 ACCGGTGCTGAGCGCGGAGGAAGGAGAAAGTCAGTCAAGGCCCCGCCACCAGTTGATCCT120 CTAGTGGAGCTCACAACTTTAGAATCGGTTCATGACGCGTTGGCGAAGGCCGAGCGACTT180 CGGAACTACTTCCAGGTAGAGCGTGACAAGGTGAATGACTTCTGGACGATTACAAAGGGG240 GAGGTGGAGACTTATCGCAATCGGCTGTTCAATGCGGAGGCGAGCATTGAAGAACTGGAG300 CGGTCACACCAGGTAGAGATGAAGGTATACAAGCAGAGGGTGCGTCACCTCATCTATGAG360 CGGAAGAAGAAGGCGCAGGCGTGCCAGGATGAAAGTCACCGTCTGCTTCGCGAGGCGGAA420 GACCGGCACCTCCAGCGCATGAATGAGATACAGGCTAAGCTCCAACAGCAAGACCAGCAG480 CTCCGGGCAGCAGCGGCTGACCATGAAATGAACGTGTACGAGAAGCGCGATTCGCACAGC540 TACATGGTAACCGTTACAAAAACACAGAGTCATGAAAAGGAGCTCGCGCGACTGCAGGTA600 TCCTGTGAGGCCAAGTTAAAAGTGTTGCGGGATGAACTGGAGTTAAGACGCCGTCGCCAG660 ATTCATGAGATTGAAGAAAGAAAGAATGAGCACATAAACGCCCTCATTAAGCAGCATGAA720 GAGAAATTTCATGAAATGAAGACATACTACAACCAAATAACCACAAATAACCTAGAAATC780 ATTCATTCCTTAAAGGAAGAAATAGCGCAGATGAAGCAGAACGACGAGCATAATGAGACT840 TTAATGTATGATATTGATCGGGAGAATCAAAATCTTGTTGCACCGTTAGAAGAAGCTCAG900 CGTGAGGTTGCGGAGCTGCAGCAGAAACGGAAGCAGAATGAACAGAACAAGCGGGGTCTC960 GAGGTCACTCGTGTTAAGTTAAGGTCGTTGCGTGAGGAGATTCGCCGACAGCGTGAAGAA1020 CATCAGGCCTTGGAGGAGCGTTACGCCTGCGTGCACCGGGAGCGCGAGGAGCTCAAGGGG1080 AAGTTTGAGTCCGCGCTCCGGCAAGCGGTGATGGTAGTCGAGGAGCGCAATGAGGTTCTC1140 CAGCAAAAGCTTATCGAGTCTCACGCTCTTGTAGAGGAAAGGGATGTACAACTTGAAGGT1200 GTTTTGCGCGCCATGAACCTCGAACCAAAGACGCTGGAACTCATCGCGACTGAGGTCGAC1260 GAATGGCTTCAACGAAAAAATCAACTGATAAAAGACTTACACTTTGAGCTTAAGAAAGGA1320 GAAAAGTTGTACAGCGCGACGTTGCTCGAGATGGAGAGCGTTGCCAGACGGCTAACATTG1380 CTTCACTGCCACGTAGCAACTTTGAGTAGGTGTTGTGGTTCACACGTTGGTTGTTCCAAG1440 TTACGGCTTTGTTGCAGCTCGCATTCGGCCGTGGGCGTGGTGGGCTGTTTTTTTTTTTCT1500 TCTGTCCTGTTGCCTCTTTCCCCTTTCTAGTGGGCCACTGCGCTTCCTATGGACCTGTGA1560 ATGTAGAACTACGCGTCACACGCCTTGGTATGTATGTTGTTACGTGCCGGATATAGAGAC1620 AGTTGCTGCTGCGACGAGCGTCGTTGTGAGACGCGTGAGTGATTGCGAGGCGAAACCTAT1680 AAAGATTGAGGCCGGTTATCATTGTAACCTCACTTTATTGTCATTTCACTAAAAAAAAAA1740 AAAAAAAAAA1750 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TATATTGACTGCATCGTGGCGTACCCCGTAGGCTCTTCTCGTTTT45 TyrIleAspCysIleValAlaTyrProValGlySerSerArgPhe 151015 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TyrIleAspCysIleValAlaTyrProValGlySerSerArgPhe 151015 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1479 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1479 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGTCACCACGGACCGGTGCTGAGCGCGGAGGAAGGAGAAAGTCAGTC48 MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 202530 AAGGCCCCGCCACCAGTTGATCCTCTAGTGGAGCTCACAACTTTAGAA96 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 354045 TCGGTTCATGACGCGTTGGCGAAGGCCGAGCGACTTCGGAACTACTTC144 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 505560 CAGGTAGAGCGTGACAAGGTGAATGACTTCTGGACGATTACAAAGGGG192 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 657075 GAGGTGGAGACTTATCGCAATCGGCTGTTCAATGCGGAGGCGAGCATT240 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 80859095 GAAGAACTGGAGCGGTCACACCAGGTAGAGATGAAGGTATACAAGCAG288 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 100105110 AGGGTGCGTCACCTCATCTATGAGCGGAAGAAGAAGGCGCAGGCGTGC336 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 115120125 CAGGATGAAAGTCACCGTCTGCTTCGCGAGGCGGAAGACCGGCACCTC384 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 130135140 CAGCGCATGAATGAGATACAGGCTAAGCTCCAACAGCAAGACCAGCAG432 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 145150155 CTCCGGGCAGCAGCGGCTGACCATGAAATGAACGTGTACGAGAAGCGC480 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 160165170175 GATTCGCACAGCTACATGGTAACCGTTACAAAAACACAGAGTCATGAA528 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 180185190 AAGGAGCTCGCGCGACTGCAGGTATCCTGTGAGGCCAAGTTAAAAGTG576 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysLeuLysVal 195200205 TTGCGGGATGAACTGGAGTTAAGACGCCGTCGCCAGATTCATGAGATT624 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 210215220 GAAGAAAGAAAGAATGAGCACATAAACGCCCTCATTAAGCAGCATGAA672 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 225230235 GAGAAATTTCATGAAATGAAGACATACTACAACCAAATAACCACAAAT720 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 240245250255 AACCTAGAAATCATTCATTCCTTAAAGGAAGAAATAGCGCAGATGAAG768 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 260265270 CAGAACGACGAGCATAATGAGACTTTAATGTATGATATTGATCGGGAG816 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspIleAspArgGlu 275280285 AATCAAAATCTTGTTGCACCGTTAGAAGAAGCTCAGCGTGAGGTTGCG864 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 290295300 GAGCTGCAGCAGAAACGGAAGCAGAATGAACAGAACAAGCGGGGTCTC912 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 305310315 GAGGTCACTCGTGTTAAGTTAAGGTCGTTGCGTGAGGAGATTCGCCGA960 GluValThrArgValLysLeuArgSerLeuArgGluGluIleArgArg 320325330335 CAGCGTGAAGAACATCAGGCCTTGGAGGAGCGTTACGCCTGCGTGCAC1008 GlnArgGluGluHisGlnAlaLeuGluGluArgTyrAlaCysValHis 340345350 CGGGAGCGCGAGGAGCTCAAGGGGAAGTTTGAGTCCGCGCTCCGGCAA1056 ArgGluArgGluGluLeuLysGlyLysPheGluSerAlaLeuArgGln 355360365 GCGGTGATGGTAGTCGAGGAGCGCAATGAGGTTCTCCAGCAAAAGCTT1104 AlaValMetValValGluGluArgAsnGluValLeuGlnGlnLysLeu 370375380 ATCGAGTCTCACGCTCTTGTAGAGGAAAGGGATGTACAACTTGAAGGT1152 IleGluSerHisAlaLeuValGluGluArgAspValGlnLeuGluGly 385390395 GTTTTGCGCGCCATGAACCTCGAACCAAAGACGCTGGAACTCATCGCG1200 ValLeuArgAlaMetAsnLeuGluProLysThrLeuGluLeuIleAla 400405410415 ACTGAGGTCGACGAATGGCTTCAACGAAAAAATCAACTGATAAAAGAC1248 ThrGluValAspGluTrpLeuGlnArgLysAsnGlnLeuIleLysAsp 420425430 TTACACTTTGAGCTTAAGAAAGGAGAAAAGTTGTACAGCGCGACGTTG1296 LeuHisPheGluLeuLysLysGlyGluLysLeuTyrSerAlaThrLeu 435440445 CTCGAGATGGAGAGCGTTGCCAGACGGCTAACATTGCTTCACTGCCAC1344 LeuGluMetGluSerValAlaArgArgLeuThrLeuLeuHisCysHis 450455460 GTAGCAACTTTGAGTAGGTGTTGTGGTTCACACGTTGGTTGTTCCAAG1392 ValAlaThrLeuSerArgCysCysGlySerHisValGlyCysSerLys 465470475 TTACGGCTTTGTTGCAGCTCGCATTCGGCCGTGGGCGTGGTGGGCTGT1440 LeuArgLeuCysCysSerSerHisSerAlaValGlyValValGlyCys 480485490495 TTTTTTTTTTCTTCTGTCCTGTTGCCTCTTTCCCCTTTC1479 PhePhePheSerSerValLeuLeuProLeuSerProPhe 500505 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 493 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 145150155160 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 165170175 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysLeuLysVal 180185190 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 195200205 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 210215220 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 225230235240 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 245250255 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspIleAspArgGlu 260265270 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 275280285 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 290295300 GluValThrArgValLysLeuArgSerLeuArgGluGluIleArgArg 305310315320 GlnArgGluGluHisGlnAlaLeuGluGluArgTyrAlaCysValHis 325330335 ArgGluArgGluGluLeuLysGlyLysPheGluSerAlaLeuArgGln 340345350 AlaValMetValValGluGluArgAsnGluValLeuGlnGlnLysLeu 355360365 IleGluSerHisAlaLeuValGluGluArgAspValGlnLeuGluGly 370375380 ValLeuArgAlaMetAsnLeuGluProLysThrLeuGluLeuIleAla 385390395400 ThrGluValAspGluTrpLeuGlnArgLysAsnGlnLeuIleLysAsp 405410415 LeuHisPheGluLeuLysLysGlyGluLysLeuTyrSerAlaThrLeu 420425430 LeuGluMetGluSerValAlaArgArgLeuThrLeuLeuHisCysHis 435440445 ValAlaThrLeuSerArgCysCysGlySerHisValGlyCysSerLys 450455460 LeuArgLeuCysCysSerSerHisSerAlaValGlyValValGlyCys 465470475480 PhePhePheSerSerValLeuLeuProLeuSerProPhe 485490 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGGGCCACTGCGCTTCCTATGGACCTG27 TrpAlaThrAlaLeuProMetAspLeu 495500 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TrpAlaThrAlaLeuProMetAspLeu 15 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AACTACGCGTCACACGCCTTGGTATGTATGTTGTTACGTGCCGGATAT48 AsnTyrAlaSerHisAlaLeuValCysMetLeuLeuArgAlaGlyTyr 10152025 AGAGACAGTTGCTGCTGCGACGAGCGTCGTTGTGAGACGCGTGAG93 ArgAspSerCysCysCysAspGluArgArgCysGluThrArgGlu 303540 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AsnTyrAlaSerHisAlaLeuValCysMetLeuLeuArgAlaGlyTyr 151015 ArgAspSerCysCysCysAspGluArgArgCysGluThrArgGlu 202530 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TTGCGAGGCGAAACCTATAAAGAT24 LeuArgGlyGluThrTyrLysAsp 35 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: LeuArgGlyGluThrTyrLysAsp 15 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGCCGGTTATCATTG15 GlyArgLeuSerLeu 10 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GlyArgLeuSerLeu 15 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..42 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCTCACTTTATTGTCATTTCACTAAAAAAAAAAAAAAAAAAA42 ProHisPheIleValIleSerLeuLysLysLysLysLysLys 1015 A43 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: ProHisPheIleValIleSerLeuLysLysLysLysLysLys 1510 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 493 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 145150155160 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 165170175 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysLeuLysVal 180185190 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 195200205 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 210215220 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 225230235240 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 245250255 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspIleAspArgGlu 260265270 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 275280285 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 290295300 GluValThrArgValLysLeuArgSerLeuArgGluGluIleArgArg 305310315320 GlnArgGluGluHisGlnAlaLeuGluGluArgTyrAlaCysValHis 325330335 ArgGluArgGluGluLeuLysGlyLysPheGluSerAlaLeuArgGln 340345350 AlaValMetValValGluGluArgAsnGluValLeuGlnGlnLysLeu 355360365 IleGluSerHisAlaLeuValGluGluArgAspValGlnLeuGluGly 370375380 ValLeuArgAlaMetAsnLeuGluProLysThrLeuGluLeuIleAla 385390395400 ThrGluValAspGluTrpLeuGlnArgLysAsnGlnLeuIleLysAsp 405410415 LeuHisPheGluLeuLysLysGlyGluLysLeuTyrSerAlaThrLeu 420425430 LeuGluMetGluSerValAlaArgArgLeuThrLeuLeuHisCysHis 435440445 ValAlaThrLeuSerArgCysCysGlySerHisValGlyCysSerLys 450455460 LeuArgLeuCysCysSerSerHisSerAlaValGlyValValGlyCys 465470475480 PhePhePheSerSerValLeuLeuProLeuSerProPhe 485490 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 467 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 145150155160 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 165170175 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysLeuLysVal 180185190 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 195200205 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 210215220 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 225230235240 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 245250255 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspLeuAspArgGlu 260265270 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 275280285 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 290295300 GluValThrArgValLysLeuArgSerLeuArgGluGluIleArgArg 305310315320 GlnArgGluGluHisGlnAlaLeuGluGluArgTyrAlaCysValHis 325330335 ArgGluArgGluGluLeuLysGlyLysPheGluSerAlaLeuArgGln 340345350 AlaValMetValValGluGluArgAsnGluValLeuGlnGlnLysLeu 355360365 IleGluSerHisAlaLeuValGluGluArgAspValGlnLeuGluGly 370375380 ValLeuArgAlaMetAsnLeuGluProLysThrLeuGluLeuIleAla 385390395400 ThrGluValAspGluTrpLeuGlnArgLysAsnGlnLeuIleLysAsp 405410415 LeuHisPheGluLeuLysLysGlyGluLysLeuTyrSerAlaThrLeu 420425430 LeuGluMetGluSerValAlaArgArgLeuThrLeuLeuHisCysHis 435440445 ValAlaThrLeuSerArgCysCysGlySerHisValGlyCysSerLys 450455460 LeuArgLeu 465 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 452 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 145150155160 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 165170175 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysLeuLysVal 180185190 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 195200205 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 210215220 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 225230235240 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 245250255 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspIleAspArgGlu 260265270 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 275280285 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 290295300 GluValThrArgValLysLeuArgSerLeuArgGluGluIleArgArg 305310315320 GlnArgGluGluHisGlnAlaLeuGluGluArgTyrAlaCysValHis 325330335 ArgGluArgGluGluLeuLysGlyLysPheGluSerAlaLeuArgGln 340345350 AlaValMetValValGluGluArgAsnGluValLeuGlnGlnLysLeu 355360365 IleGluSerHisAlaLeuValGluGluArgAspValGlnLeuGluGly 370375380 ValLeuArgAlaMetAsnLeuGluProLysThrLeuGluLeuIleAla 385390395400 ThrGluValAspGluTrpLeuGlnArgLysAsnGlnLeuIleLysAsp 405410415 LeuHisPheGluLeuLysLysGlyGluLysLeuTyrSerAlaThrLeu 420425430 LeuGluMetGluSerValAlaArgArgLeuThrLeuLeuHisCysHis 435440445 ValAlaThrLeu 450 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 311 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 LeuArgAlaAlaAlaAlaAspHisGluMetAsnValTyrGluLysArg 145150155160 AspSerHisSerTyrMetValThrValThrLysThrGlnSerHisGlu 165170175 LysGluLeuAlaArgLeuGlnValSerCysGluAlaLysIleLysVal 180185190 LeuArgAspGluLeuGluLeuArgArgArgArgGlnIleHisGluIle 195200205 GluGluArgLysAsnGluHisIleAsnAlaLeuIleLysGlnHisGlu 210215220 GluLysPheHisGluMetLysThrTyrTyrAsnGlnIleThrThrAsn 225230235240 AsnLeuGluIleIleHisSerLeuLysGluGluIleAlaGlnMetLys 245250255 GlnAsnAspGluHisAsnGluThrLeuMetTyrAspIleAspArgGlu 260265270 AsnGlnAsnLeuValAlaProLeuGluGluAlaGlnArgGluValAla 275280285 GluLeuGlnGlnLysArgLysGlnAsnGluGlnAsnLysArgGlyLeu 290295300 GluValThrArgValLysLeu 305310 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 145 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: TLTF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: MetSerProArgThrGlyAlaGluArgGlyGlyArgArgLysSerVal 151015 LysAlaProProProValAspProLeuValGluLeuThrThrLeuGlu 202530 SerValHisAspAlaLeuAlaLysAlaGluArgLeuArgAsnTyrPhe 354045 GlnValGluArgAspLysValAsnAspPheTrpThrIleThrLysGly 505560 GluValGluThrTyrArgAsnArgLeuPheAsnAlaGluAlaSerIle 65707580 GluGluLeuGluArgSerHisGlnValGluMetLysValTyrLysGln 859095 ArgValArgHisLeuIleTyrGluArgLysLysLysAlaGlnAlaCys 100105110 GlnAspGluSerHisArgLeuLeuArgGluAlaGluAspArgHisLeu 115120125 GlnArgMetAsnGluIleGlnAlaLysLeuGlnGlnGlnAspGlnGln 130135140 Leu 145 __________________________________________________________________________

## Claims

1. A truncated lymphocyte stimulating factor polypeptide that, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation, wherein said polypeptide consists of amino acids 1 to 467 of SEQ. ID. NO: 17 or a fragment of amino acids 1 to 467 of SEQ. ID. NO. 17 possessing said in vivo activity.

2. A truncated lymphocyte stimulating factor polypeptide according to claim 1, which consists of amino acids 1 to 467 of SEQ. ID. NO. 17.

3. The truncated lymphocyte stimulating factor polypeptide according to claim 1, wherein said fragment comprises at least 150 contiguous amino acids of SEQ. ID. NO. 17.

4. A truncated lymphocyte stimulating factor polypeptide according to claim 1, which comprises at least 100 contiguous amino acids of SEQ. ID. NO. 17.

5. A truncated lymphocyte stimulating factor polypeptide according to claim 1, which comprises at least 120 contiguous amino acids of SEQ. ID. NO. 17.

6. A truncated lymphocyte stimulating factor polypeptide that, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation, wherein said polypeptide consists of amino acids 1 to 311 of SEQ. ID. NO. 19 or a fragment of amino acids 1 to 311 of SEQ. ID. NO. 19 which, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation.

7. A truncated lymphocyte stimulating factor according to claim 6, which consists of amino acids 1 to 311 of SEQ. ID. NO. 19.

8. A truncated lymphocyte stimulating factor that, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation which consists of amino acids 1 to 452 of SEQ. ID. NO. 18 or a fragment of amino acids 1 to 452 of SEQ. ID. NO. 18, which, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation.

9. A truncated lymphocyte stimulating factor according to claim 8, which consists of amino acids 1 to 452 of SEQ. ID. NO. 18.

10. A truncated lymphocyte stimulating factor polypeptide that, upon in vivo administration in a mammal, stimulates CD8+ T-cells to release gamma interferon, thereby resulting in immunosuppression or immunostimulation, wherein said polypeptide consists of amino acids 1 to 145 of SEQ. ID. NO. 20 or a fragment of amino acids 1 to 145 of SEQ. ID. NO. 20 possessing said in vivo activity.

11. A truncated lymphocyte stimulating factor polypeptide according to claim 10, wherein the polypeptide consists of amino acids 1 to 145 of said SEQ. ID. NO. 20.

12. A pharmaceutical composition containing a polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition containing a polypeptide according to claim 2 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition containing a polypeptide according to claim 3 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition containing a polypeptide according to claim 4 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition containing a polypeptide according to claim 5 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition containing a polypeptide according to claim 6 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition containing a polypeptide according to claim 7 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition containing a polypeptide according to claim 8 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition containing a polypeptide according to claim 9 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition containing a polypeptide according to claim 10 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition containing a polypeptide according to claim 11 and a pharmaceutically acceptable carrier.