SE444687B - POLYPEPTIDES WITH THE ABILITY TO INDUCE DIFFERENTIALIZATION OF BOTH TH-1? 72 + T-Lymphocytes AND BU-1? 72+ B-Lymphocytes - Google Patents

Description

c 10 15 20 25 30 35 7812614-1 2 stem cells originating in hemopathic tissues, Goldstein et al., The Human Thymus, Heínemann, London, 1969. Lymphocytes are differentiated in the thymus and released from there as mature thymus-derived cells, called T- cells, which circulate to the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation in the thymus appears to be mediated by secretions of the epithelial cells in the thymus. V It has been known for some time that thymus has been associated with the immune properties of the body and great interest has therefore been shown in the substances isolated from thymus. In this context, in recent years a relatively large number of articles have been published based on scientific work relating to materials present in thymus from cattle. A number of articles relating to research in this field can be found, for example, as follows: The Lancet, July 20, 1968, pages 119-122; Triangle ,. volume II, No. 1, page 7-lä, 1972; Annals of the New York Academy of Sciences, Volume 183, pp. 230-2HO, 1971; and Clinical and Experimental Immunology, Volume 4, No. 2, pp. 181-189, 1969; Nature, Volume 247, Page 11, 1974; Proceedings of the National Academy of Sciences USA, Volume 71, pp. 1U7H-1A78, l97Å; Cell, Volume 5, pages 361-365 and 367-370, 1975; Lancet, Volume 2, pp. 256-259, 1975; Proceedings of the National Academy of Sciences USA, Volume 72, pp. 11-15, 1975; Biochemistry, Volume 1, pp. 221H-2218, 1974; Nature, Volume 255, page N23-NZH, 1975.

A second class of lymphocytes with immune function are B-1 lymphocytes or B-cells. These are differentiated in the bursa Fabricii in birds and in an as yet unidentified organ in mammals. T cells and B cells interact in many immune respects. This is evident, for example, from articles in Science, 193, 319 (July 23, 1976) and the Cold Spring Harbor Symposia on Quantitative Biology, Volume XLI, 5 (1977).

A nonapeptide material has recently been isolated from piglets by J. F. Bach et al. and identified as "facteur thymique serique" (FTS). The insulation of this material and its structure. described in C. R. Acad. Sc. Paris, p. 283 (November 29, 1976), Series D-1605 and Nature 266, 55 (March 3, 1977). The structure of this nonapeptide has been identified as GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN, wherein "GLX" denotes either glutamine or pyro- The material in which GLX is glutamine or pyroglu- In these articles, Bach shows that åzwzf fi ln. _. . glutamic acid. tamic acid has been synthesized. _ ... vi waí -... s fi q-fvff- fl-. fw- "M 10 f Yö1Zb14" 1 * 3 the nonapeptide FTS selectively differentiates T cells (and not B cells) by using an E- rosette analysis. Bach therefore concluded that the material was a thymic hormone. a more careful examination of the activity of this nonapeptide showed that FTS differentiated both T cells and B cells and therefore had an activity more similar to ubiquitin than thymopoeitin, Brand, Gilmor and Goldstein, Natur, 259, 597 (1977).

It has now been found that a synthesized U-amino acid polypeptide segment of this FTS nonapeptide has many of the properties of the nonapeptide described in the above publications.

Accordingly, an object of the present invention is to provide novel polypeptide segments and polypeptides that are biologically important.

Another object of the invention is to provide novel polypeptide segments and polypeptides which are capable of inducing differentiation of both T lymphocytes and B lymphocytes and are therefore very useful in the immune system of humans and animals.

A further object of the invention is to provide methods for synthesizing new polypeptide segments and polypeptides according to the invention. Other objects and advantages of the invention will become apparent from the following description.

According to the invention, a novel biologically active polypeptide segment is obtained having the following amino acid sequence: -ALA-LYS-SER-GLN-.

The biological activity of this polypeptide segment is generally maintained upon substitution of either one or both of: 1) L-alanyl at position 1; and 2) L-seryl at position 5 with a natural or non-natural amino acid residue. substituted polypeptide segments are remarkably potent.

Recently, some of these Terminal substitutions of the present polypeptide segments yield the present polypeptides.

Also shown herein is a method of preparing the polypeptide segments and polypeptides of the invention by solid phase peptide synthesis, as well as therapeutic compositions containing the polypeptides and methods of administering them to humans and animals to produce a biological effect.

As stated above, the present invention relates to novel polypeptide segments and polypeptides of therapeutic value in various fields, and methods of using them using the polypeptides of the invention, and methods of making these.

According to an essential embodiment of the invention, a biologically active polypeptide segment is obtained which has the following amino acid sequence: I. -ALA-LYS-SER-GLN-.

Since the biological activity of this polypeptide segment is generally conserved, then a natural or non-natural amino acid residue may replace either or both of: l) alanyl at position 1; and 2) seryl at position 3, this embodiment of the invention further comprises biologically active polypeptide segments having the following amino acid sequence: 1A. -X-LYS-Y-GLN- wherein X and Y can each be selected from the group consisting of natural and unnatural α-aminocarboxylic acid (hereinafter "amino acid") residues. Although it is likely that the vast majority of such substitutions in groups X and Y allow for conserved biological activity, it is possible that some natural or unnatural amino acid residues will affect the folding of the molecule (which will be discussed in more detail below ) and thereby substantially eliminate the biological activity.

Such activity-destroying substituents are not encompassed by the present invention. In more detail, X represents sarcosyl, L-alanyl or D-alanyl, Y represents seryl, sarcosyl-2-methyl-alanyl or D-alanyl.

If a particular substitution allows the biological activity of the polypeptide to be preserved, it can be readily determined by testing its differentiation of Th-1 + T-lymph0GyC @ P °° h BU'l B lymphocytes in the chicken induction assay described below.

Compounds that are particularly active in concentrations of ng / ml (approximately 1 ng / ml or less) in this assay are considered to be biologically active. A list of natural amino acids can be found in many manuals, such as RT Morrison and RN Boyd »" 0P8aniC Ch? MiStry "* Allyn and Bacøn, 1959, Chapter 33. In addition to the natural amino acids (which are those found in proteins), There are also a number of so-called "unnatural" amino acids which have not been found in proteins, although they sometimes occur in nature as metabolic intermediates or the like. These non-natural amino acids may be D-isomers corresponding to the optically active (L-form) natural amino acids or they may be completely different chemical compounds such as sarcosine (N-methylglycine) or 2-methylalanine as indicated above. Lists of such non-natural amino acids are also found in many reference works. In addition, the polypeptide segment set forth in the above embodiment of formula IA must contain terminal substituents on the 4-amino acid sequence in order to obtain the present polypeptides. These terminal substituents must not significantly affect the biological activity of the active 4-amino acid segment, measured as the ability to induce differentiation of Th-1 + T lymphocytes and Bu-1 + B lymphocytes in the chicken induction assay described below. The present polypeptides can be described by the general formula: II. RX-LYS-Y-GLN-R 'A wherein X and Y have the meaning given above and R and R' represent substituents on the terminal amino group and on the terminal carboxyl group of the peptide segment, respectively, which, as stated above, do not significantly affect the biological activity of the active amino acid segment. Since the active tetrapeptide segment is present in a longer sequence in the naturally occurring material isolated by Bach, it is clear that the terminal amino and carboxylic acid groups are not essential for the biological activity of the tetrapeptide segment, as is the case for certain polypeptides. The present invention therefore includes not only those tetrapeptide segments which are substituted by H and OH, respectively, but also those which are terminally substituted by one or more other functional groups which do not substantially affect the biological activity described herein. It should be noted, however, that the nonapeptide described by Bach et al. specifically, is excluded from the scope of the present invention.

It can be seen that these functional groups include a normal substitution such as acylation on the free amino group and amidation of a free carboxylic acid group, as well as substitution of other amino acids and polypeptides. In these respects, the polypeptide segments of the invention appear to be very unusual as they exhibit the same biological activity as the natural nonapep. ~ - »r- KH 10 20 25 50 7812614-1 time of which the active tetrapeptide segment forms part. It is therefore believed that the activity requirements of the molecule are achieved through its Hero stereochemistry, i.e. the special "folding" of the molecule. It should be noted that the polypeptide bonds are not rigid but flexible, and polypeptides may be in planar form, such as helices and the like. This means that the whole molecule is flexible and can be "folded" in a number of ways. In the present invention, it has been found that the novel tetrapeptide segments are likely to be "folded" in a similar manner to the corresponding tetrapeptide segments in the natural non-peptide by exhibiting the same biological properties. For this reason, the tetrapeptide segments may be terminally substituted with different functional groups provided that the substituents do not substantially affect the biological activity or affect the natural "fold" of the molecule. A The ability of the molecule to preserve its biological and natural fold is clear from the fact that the tetrapeptide segments of the invention exhibit the same biological properties as the natural 9-amino acid peptide described as FTS by J. F. Bach in the above articles. In this nonapeptide, a tetrapeptide sequence according to the invention can be identified in the molecule but only in combination with the other amino acids described therein. Since the tetrapeptide segments of the invention provide the same biological activity as the nonapeptide FTS, it is clear that the amino acids and peptide chains substituted on the terminal amino acid residues of the tetrapeptide segment do not affect its biological properties.

R and R 'in formula II thus represent a substituent which does not substantially affect the biological activity of the active segment. R and R 'represent any of the following substituents: 1.Al in hydrogen os in NH 2 M * However, as pointed out above, R may also be neutral amino acid groups. An example may be mentioned: 7812614-1 B H ~ GLN H-SAR A preferred embodiment of the invention is that in which X represents L-alanyl, Y represents L-seryl, R represents hydrogen and R 'represents OH. This preferred embodiment can be chemically designated as follows: V H2N-çH-CONH-CH-CONH-çH-CONH-CH-COOH II cH¿ (cH2) u cnz NH2 OH CONH2 H-ALA »Lyssaa- GLN A second preferred embodiment is that wherein X and Y each represent a group consisting of sarcosyl or D-alanyl or Y represents 2-methyl-alanyl, preferably X represents sarcosyl, Y sarcosyl or D-alanyl , R is hydrogen and R 'is NH2.

The invention also encompasses pharmaceutically acceptable salts of the polypeptides. As acids which can form salts with the polypeptides there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid etc. and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid or sulfanilic acid.

Throughout this application, the amino acid components of the peptide are identified by abbreviations. These abbreviations are given below. D-amino acids are indicated by placing "D" before the abbreviation, for example D-alanine is denoted by "D-ALA".

Amino acid Abbreviated designation L-alanine ALA Lfserin SER L-glutamine GLN L-lysine LYS L-valine Val Sarkosin SAR 2-methylalanine 2-Me-ALA Skin 7812614-1 The polypeptides of the invention are U-amino acid peptides (and substituted derivatives thereof) which have been found to exhibit similar properties to the 9-amino acid polypeptide FTS isolated from pig blood as described in the above-referenced articles by Bach et al. The peptides of the invention are particularly characterized by their ability to induce differentiation of T-mother cells as well as B-mother cells. Some of the present polypeptides are active at as low a concentration as one picogram (pg) / ml in the chicken induction assay described below. It has been found that the polypeptides of the invention induce differentiation of immunocyte parent cells in vitro in the same manner as the nonapeptides described by Bach. Thus, the polypeptides of the present invention have been shown to induce differentiation of both T mother cells, measured by the formation of thymic differentiation antigen Th-1, and B mother cells, measured by the formation of differentiation antigen Bu-1. In other words, the present polypeptides are capable of inducing differentiation of both Th-1 + T lymphocytes and Bu-1 * B lymphocytes.

It has also been shown that the present polypeptides increase the ability of in vivo production of cytotoxic lymphocytes upon administration of the present production of cyto-vitro assay of cell cytotoxic lymphocymulation with allogeneic antigens. Thus, pølypeptides to, for example, rats produce a toxic lymphocyte precursor measured by a rat spleen. Since the generation of ter directly corresponds to the degree of inoculation at an allogeneic inoculation vs. host reaction, the above results provide further support for the immunological utility of the present polypeptides.

In order to explain the importance of the differentiating biological properties of the polypeptides according to the invention, it should be noted that the function of the thymus in relation to immunity can generally be stated as the production of thymus-derived cells or lymphocytes, called T cells. T cells make up a large proportion of all circulating small lymphocytes. The T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homoymp responses), such as effector cells. However, T cells do not secrete humoral antibodies. These antibodies are secreted by cells (designated B cells) derived directly from the bone marrow regardless of thymus influence. However, for many antigens, B cells require the presence of appropriately reactive T cells. cells before they can produce antibodies. The mechanism of this cell interaction is not yet fully understood.

This shows that the thymus is necessary for the development of cellular immunity and many humoral antibody responses and affects these systems by inducing, within the thymus, the differentiation of hemopoknial stem cells into T cells. This inductive influence is mediated by secretions from the epithelial cells in the thymus, ie thymic hormones. 'A To understand the function of the thymus and the cell system of lymphocytes and the circulation of lymphocytes in the body, it should also be pointed out that stem cells arise in the bone marrow and when the thymus via the bloodstream. Within the thymus, the stem cells are differentiated into immunologically competent T cells, which migrate to the bloodstream and, together with B cells, circulate between tissues, lymph vessels and the bloodstream.

The cells in the body that secrete antibodies (B cells) also develop from homeopathic stem cells, but their differentiation in birds takes place. In mammals, thymus is not determined. in an organ analogous to the thymus, called the bursa Fabriciáu. animals, no equivalent organ has been found and it is believed that these cells differentiate within the bone marrow. They are consequently called bone marrow-derived cells or B-cells. The physiological substances that dictate this differentiation are completely unknown.

As noted above, the polypeptides of the present invention are therapeutically useful for the treatment of humans and animals. Because the novel polypeptides have the ability to induce differentiation of lymphophoric stem cells derived from hemopmatic tissue into both thymus-derived lymphocytes (T cells) and immunocompetent B cells that can interfere with the body's immune response, the products of the present invention are considered to have a multiple therapeutic use.

Because the compounds have the ability to perform some of the indicated functions of the thymus, they can primarily be used in various areas of thymic function and immunity. A primary use is the treatment of DiGeorge syndrome, a condition in which there is a born lack of thymus. Injection of one of the present polypeptides, as detailed below, overcomes this deficiency.

Another area of application is agammaglobulinemia, which is due to a defect in the probable B-cell differentiating hormone in the body.

Injection of one of the present polypeptides overcomes this deficiency. Because the present polypeptides are extremely active in low concentrations, they are useful for enhancing the collective thinking immunity of the body by increasing therapeutic stimulation of cellular immunity and humoral immunity and thereby are useful for the treatment of diseases involving chronic infections in vivo, such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like. The present peptides are further considered useful in any field where cellular or humoral immunity is a remedy and especially when there are deficiencies in immunity such as in the DiGeorge syndrome indicated above. Due to the properties of the polypeptides, they are further useful in vitro to induce the development of surface antigens by T cells, to induce the development of the functional capacity to achieve susceptibility to mitogens and antigens, and cell interaction to increase the ability of T cells. B cells to produce antibodies. They are useful in vitro for inducing the development of B cells measured by the development of surface receptors for complement. The present peptides are also useful for inhibiting an uncontrolled DP01Üï fl Etion of lymphocytes that can be affected by ubiquitin (US-PS N 002 602).

An important property of the present polypeptides is their ability in vivo to restore cells with the properties of T cells and also their ability in vivo to restore cells with the properties of B cells. They are therefore useful for the treatment of relative or absolute B-cell deficiencies as well as relative or absolute T-cell deficiencies, whether these deficiencies are due to deficiencies in the tissue that differentiates B-cells and thymus, respectively, or has some other abnormality.

Another important property of polypeptides according to the invention is that they are highly active in very low concentrations. Thus, it has been found that the polypeptides in general are active at concentrations of about 1 ng / ml, while some surprisingly potent polypeptides (H-SAR-LYS-D-ALA -GLN- NH2 and H-SAR-LYS-SAR-GLN-NH2) are active in concentrations ranging from about 0.1 pg / ml. The carrier may be any well known carrier for this purpose including normal saline solutions, suitably with a protein diluent such as bovine serum albumin to prevent adsorption losses to the glass at these low concentrations. The polypeptides of the invention are generally active in a range above about 1 pg / kg body weight, while some very potent polypeptides are active from about 1 ng / kg body weight. For the treatment of DiGeorge syndrome, the polypeptides may be administered in an amount of about 1 to about 100 pg / kg body weight, while the very potent polypeptides may be administered in an amount of about 1 to 100 ng / kg. the same dose range is used for the above body weight. treatment of other specified conditions or diseases. they apply to parenteral administration, but it should also be noted that oral administration is possible in dose ranges which are generally about 100 to 1000 times greater than for injection.

The polypeptides of the invention were prepared in a manner similar to that described by Merrifield in the Journal of the American Chemical Society, 85, pp. 219-215U, 1965. The synthesis involved a stepwise addition of protected amino acids to a growing peptide chain bound by covalent bonds to a solid resin particle. By this method, reagents and by-products could be removed by filtration and recrystallization of intermediates could be eliminated. The basic idea of this method is an attachment of the C-terminal amino acid in the chain to a solid polymer by a covalent bonding and addition of subsequent amino acids one at a time step by step until the desired sequence is assembled. The peptide is finally removed from the solid support and the protecting groups are removed. This method provides a growing peptide chain attached to a completely insoluble solid particle so that it is in a convenient form for filtration and purification from reagents and by-products. The amino acids can be attached to any suitable polymer as a; must be able to be separated from reagents that have not reacted easily. The polymer may be insoluble in used solvents or may be soluble in some solvents and insoluble in others.

The polymer should have a stable physical form which allows for easy filtration. It must contain a functional group to which the first protected amino acid can be firmly bound by a covalent bond. Various insoluble polymers suitable for this purpose are those such as cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, but in the synthesis of the invention a chloromethylated copolymer of styrene and divinylbenzene is generally used.

Polymers that are soluble in organic solvents and insoluble in aqueous solvents can also be used. One such polymer is a polyethylene / glycol having a molecular weight of about 20,000, which is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide synthesis is described in F. Bayer and M. Mutter, Nature »257, 512 (1972) and references cited therein.

The various functional groups in the amino acid that were active but would not participate in the reactions were protected with conventional protecting groups used in the polypeptide technique throughout the reaction. The functional group on lysine was thus protected with protecting groups that could be removed when the sequence was complete without adversely affecting the finished polypeptide product. In the synthesis, fluorescamine was used to determine if the coupling was complete by an indication of positive fluorescence (see Felix et al., Analyt. Biochem., 52, 577, 1973). If a complete coupling was not indicated, the coupling was repeated with the same protected amino acid before the protection was removed.

The C-terminal amino acid can be attached to the polymer in a number of different well known ways. Summaries of halomethyl resin adhesion methods are set forth in Horiki et al., Chem Letters, pp. 165-168 (1978) and Gisin, Helv. Chim. Acta, 56, 1476 (1973) As well as references cited therein.

The process generally comprised a first esterification of L-glutamine, with protected amino groups, to the absolute alcohol resin containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and dried.

The protecting group on the α-amino group of the glutamine amino acid (eg V t-BOC, ie t-butyloxycarbonyl) was then removed. The resulting coupled amino acid resin, with a free amino group, was then reacted with a protected L-serine, preferably α-t-BOC-Ä-O-benzyl-L-serine to couple L-serine. The reactions were then repeated with protected L-lysine and L-alanine until a complete molecule was prepared. «The reaction sequence was performed as follows; , ._._.

U. 7812614-1 13v resin L a-R1-Gln-OH Q-Rlecin-narcs I q-amino-protecting group {removed V H-Gln-resin R 2 § II -R -L-Ser-OH el ”q-R1- Ser-Gln resin c-amino protecting group R is removed 12V H-Ser-Gln resin 'R; IB ¿Q-R1-Lys-OH R. R "» 15 x2 -R1 ~ Lys-Ser-Gln resin u-amino protecting group is removed R. 15 É20 H-Lys-Ser-Gln resin IVL Q-al-Ala -ou É3 V32 α-R1-Ala-Lys-wound-cin-have g all protecting groups and the resin is removed v H-Ala-Lys-Ser-Gln-OH In the above reaction sequence, R1 represents a protecting group for the α-amino group and R2 and R5 denotes protecting groups for the reactive side chains of L-serine and L-lysine, respectively, which are not affected or removed when R1 is removed to enable a further reaction.

In the above pentapeptide resin, the term R1 suitably denotes a protecting group such as t-butyloxycarbonyl, R2 denotes benzyl and H3 denotes substituted benzyloxycarbonyl (eg 2,6-dichloro-benzyloxycarbonyl) above as being useful in the process. the final intermediate was prepared, the peptide resin was cleaved to remove protecting groups R1, R2 and RB thereon and the resin or substituted benzyl (eg H-chlorobenzyl) I: The resin is any of the resins indicated. The protecting groups were removed at some time. 7812614-1 in a conventional manner, for example by treatment with anhydrous hydrogen fluoride, and the resulting free peptide was subsequently recovered.

As noted above, in carrying out the process, it is necessary to protect or block the amino groups in order to control the reaction and obtain the desired products. Suitable amino protecting groups that can be used include salt formation to protect strongly basic amino groups, or urethane protecting substitutes such as p-methoxybenzyloxycarbonyl and t-butyloxycarbonyl. It is convenient to use t-butyloxycarbonyl (BOC) or t-amyloxycarbonyl (AOC) to protect the α-amino group of amino acids which react at the carboxyl terminus of the molecule, since BOC and AOC (t-amyloxycarbonyl) protecting groups can be easily removed after such a reaction and before subsequent steps (in which such a u-amino group itself reacts) by a relatively mild action of acids (eg trifluoroacetic acid), which treatment does not otherwise affect groups used to protect other reactive side chains. Thus, it should be noted that α-amino groups can be protected by reaction with any material which can protect the amino groups for subsequent reaction (s) but which can later be removed under conditions which do not otherwise affect the molecule. Examples of such materials are organic carboxylic acid derivatives which acylate the amino group.

In general, each amino group can be protected by reaction with a compound containing a grouping of the formula: O u Ru-o-c- wherein RM represents a grouping which prevents the amino group from participating in subsequent coupling reactions and which can be removed without destroying the molecule. Ru thus represents a straight or branched alkyl group which may be unsaturated, suitably having 10 to 10 carbon atoms, and preferably substituted by halo or cyano; aryl, preferably with 6-15 carbons; cycloalkyl, preferably having 5-8 carbon atoms; aralkyl, preferably having 7-18 carbon atoms; alkaryl, suitably having 7 to 18 carbon atoms; or a heterocyclic group, for example isonicotinyl. The aryl, aralkyl and alkaryl groups may also be further substituted as with one or more alkyl groups having 1 to about 4 carbon atoms. Preferred groups for R include t-butyl, t-amyl, tolyl, xylyl and benzyl. Highly preferred specific amino protecting groups include benzyloxycarbonyl; substituted benzyloxycarbonyl, wherein the phenyl ring is substituted with one or more halogens, eg Cl or Br; nitro; lower alkoxy, eg methoxy; lower alkyl; t-butyloxycarbonyl; t-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; biphenylisopropoxycarbonyl and the like. Other protecting groups that may be used include isonicotinyloxycarbonyl, phthalyl, p-tolylsulfonyl, formyl and the like. In carrying out the general procedure of the invention, the peptide is built up by reaction between the free u-amino group and a compound having protected amino groups. For reaction or coupling, a compound to be attacked at its carboxyl group is activated so that the carboxyl group can then react with the free α-amino group on the attached peptide chain. To effect activation, the carboxyl group may be converted to any reactive group such as an ester, anhydride, azide, acid chloride or the like. Alternatively, a suitable coupling reagent may be added during the reaction. Suitable coupling reagents are described, for example, in Bodanszky et al., - Peptide Synthesis, Interscience, Second Edition, 1976, Chapter 5, comprising carbodiimides (eg dicyclocarbodiimide), carbonyldiimidizole and the like.

It should also be noted that during these reactions the amino acid groups contain both amino groups and carboxyl groups and usually one grouping reacts while the other is protected. Prior to the coupling step, the protecting group on the α- or terminal amino group of the affected peptide is removed under conditions that do not significantly affect other protecting groups, such as the group on epsilon-amino in the lysine molecule. A preferred method of performing this step is mild acidolysis, such as reaction at room temperature with trifluoroacetic acid. A The process steps described above give, as can be seen, a tetrapeptide of formula III: III. H-ALA-LYS-SER ~ GLN-OH This tetrapeptide contains a tetrapeptide segment according to the invention which is necessary for biological activity.

Either or both of L-alanyl and L-seryl can be replaced by a natural or non-natural amino acid residue by replacing either one or both of alanine γ and serine with an appropriately protected natural or non-natural amino acid in the above synthetic scheme. , whereby a tetrapeptide of the following formula is obtained: IIIA. H-X-LYS-Y-GLN-OH wherein X and Y are as defined above. The substituted tetrapeptide of formula II, wherein the terminal amino acid groups may be further substituted as indicated above, may then be prepared by reacting the tetrapeptide of formula ( IIIA) or the protected peptide resin starting compound with suitable reagents for the preparation of desired derivatives. Reactions of esterification, amidation and the like Furthermore, other amino- types, such as acylation, are of course well known in the art. acids, i.e. amino acid groups that do not affect the biological activity of the tetrapeptide sequence, are added to either end of the peptide chain by the same reaction sequence according to which the tetrapeptide itself was synthesized. Furthermore, a replacement of alanine and / or serine can be achieved by using the desired substituent (suitably protected) instead of alanine or serine in the previous reaction sequence in which the unsubstituted tetrapeptide was synthesized. Although the Merrifield solid phase technique has been used to prepare the present polypeptides, it is clear that classical techniques as described, for example, by M. Bodanszky and M. A.

Ondetti in Peptide Synthesis, Interscience, 1966 can also be used. With respect to the nonapeptides described according to Bach, the latter teaches that the site of biological activity is on the C 'terminal part, which is completely contrary to the present invention. In addition, certain remarkably strong peptides of the present invention are active at concentrations much lower than the PTS nonapeptide itself. This result is totally unexpected and can in no way be predicted. The useful dosage range for FTS is described to be from 0.0001 to about 1.0 mg / kg body weight (0.1 pg / kg - 1.0 mg / kg). In contrast, the remarkably strong peptides of the present invention were found to be active from about 1 ng / kg body weight, 100 times less than the lower value of the range given in column 9 of Bach's U.S. Patent N 1H8,886 (which describes same subject as the Nature publication). In this context, it is obvious that the synthetic preparation of the present U-linked peptide is unconditionally advantageous compared to utilizing a pig room, such as that described by Bach (ie synthetic preparation of a compound enables a clean as well as safe supply to be provided. , compared to the impure and unsafe supply, when a material must be obtained from an animal). In addition, if Bach's nonapeptide were to be synthesized, the present tetrapeptide would cost considerably more. BO 55 7812614-1 The identity and purity of the present peptides were determined according to such well known methods as thin layer chromatography, electrophoresis, amino acid analysis and the like.

The invention is further illustrated by the following examples, in which parts are by weight unless otherwise indicated.

Example I For the preparation of a polypeptide according to the invention the following materials were obtained: q-BOC-L-glutamine-o-nitrophenyl ester α-BOC-E-2-chloro-benzyloxycarbonyl-L-lysine α-BOC-O-benzyl -L-serine and ~ BOC-L-alanine.

In these reagents, BOC denotes t-butyloxycarbonyl. of "Sequenal" grade for amino acid sequencing, dicyclohexylcarbodiimide, fluorescamine and the resin were also commercially available. The resin used was a polystyrene divinylbenzene resin having a size of 200-H00 mesh containing 1 *% divinylbenzene and 0.75 mM chloride per g of resin.

To prepare the polypeptide, 2 mmol of α-BOC-L-glutamine was esterified to 2-mmol of chloromethylated resin in absolute alcohol containing 1 mM triethylamine for 2H hours at 80 ° C. The resulting amino acid resin ester was filtered, washed with absolute alcohol and dried. Thereafter, the other d-BOC amino acids were similarly coupled to the deprotected u-amino group of the peptide resin in the correct sequence so that the polypeptide of the invention was obtained using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, a portion of the resin was tested with fluorescamine, and if a positive fluorescence could be detected, the coupling was considered incomplete and repeated with the same protected amino acid. As a result of these coupling reactions, the tetrapeptide resin was prepared as an intermediate.

This peptide-resin was cleaved and the protecting groups were removed in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 ° C for 60 minutes with 1.2 ml of anisole per g of peptide-resin as scavenger. The peptide mixture was washed with anhydrous ether and extracted with aqueous acid.

The extract was lyophilized and the peptide was chromatographed on P-6 Bio-Gel in 1 N acetic acid. The resulting polypeptide was determined to be 9N% pure and to have the following composition: Reagent 10 H 2 ALA-LYS-SER-GLN-OH For identification, thin layer chromatography and electrophoresis were performed as follows. A thin layer chromatography was performed on a 30 μg pbov_silica gel (Brinkman Silica gel with fluorescent indicator, 20 X 20 cm, thickness 0.1 mm) with the following eluent: Rfšz n-butanol: pyridineacetic acid: water; 30: l5: 3: l2 Rf: ethyl acetate: pyridineacetic acid zvatteng 5: 5: l: 3 Rfšz ethyl acetate: n-butanolzacetic acid zvatteng 1: 1: 1: l Electrophoresis was performed on a 100 μg sample on whatman paper 3 mm (ll, 5 x 56 , 5 cm) using a pyridine acetate buffer solution with a pH of 5.6 and a voltage of 1000 V for 1 hour.

Spray reagents for both thin layer chromatography and electrophoresis were Pauly and ninhydrin. The following results were obtained: Rfl = immobile, Rfz = immobile and Rfj = 0.336. Electrophoresis gave a displacement of 9, U cm towards the cathode.

Example II To determine the activity and properties of the tetrapeptide product prepared in Example I, the following chicken induction assay was used. This analysis is described in more detail in Brand et al., Science, 193, 319-321 (July 25, 1976) and references cited therein. g Bone marrow from newly hatched chickens was chosen as a source of inducible cells due to its lack of a larger number of Bu-li or Th-1 + cells. Polar cells from the femur and tibiotarsus of five newly hatched chickens of the strain SC (Hy line) were fractionated by ultracentrifugation on a five-layer discontinuous gradient of bovine serum albumin (BSA). Cells from the two lightest layers were pooled, washed and suspended for incubation at a concentration of 5 x 10 6 cells / ml with an appropriate concentration of the test polypeptide in RPMI 1630 medium supplemented with 15 mM hepes, 5% gamma globulin-free feta fi in calf serum, deoxyribonuclease ( 14 to 18 units / ml), heparin (5 units / ml), penicillin (100 units / ml) and streptomycin (100 ug / ml). Controls were incubated with BSA (1 μg / ml) or medium alone. After the incubation, the cells were tested in the cytotoxicity assay using chicken C1 and guinea pigs C2 to C9 complement fractions as described in the reference article. The amount of Bu-1 * or Th-1 + cells in each layer was calculated as a cytotoxicity index, 100 (a-b) / a, 10 nß 19 7812614-1 wherein a and b denote the percentage of viable cells in the complement control and the test preparation, respectively. The percentage of cells induced was obtained by subtracting mean values in the control incubations without inducers (usually 1 to 5%) from those for the test inductions.

The specificity of the effect of the test polypeptide and its similarity to ubiquitin was demonstrated by inhibiting the induction of Bu-1 + B cells and Th-1 + T ~ cells by the test polypeptide upon addition of ubiquitin at a concentration of 100 pg / ml. This high dose of ubiquitin inactivates the ubiquitin receptors, thereby preventing the induction of cells by any agent acting through these receptors. As a result of this analysis, it was found that the tetrapeptide of Example I showed a biological activity similar to that of ubiquitin in inducing differentiation of both Th-1 + and Bu-1 * B lymphocytes in ng / ml concentrations.

Example III A. The assay of Example II was repeated using as test polypeptide one of the following: H-GLN-ALA-LYS-SER-GLN-OH H-SAR-ALA-LYS-SER-GLN-OH In each case, a biological activity similar to that of ubiquitin is observed.

B. The assay of Example II was repeated, using as test polypeptide one of the following: H-SAR-LYS-D-ALA-GLN-NH2 H-SAR-LYS-SAR-GLN-NH2 H-D-ALA-LYS- In all cases, a biological activity similar to that of ubiquitin could be observed in D-ALA-GLN-NH2 VI. For the first of these polypeptides, this activity was observed in a concentration range from about 1 pg / ml to about 100 pg / ml. at a concentration as low as 0.1 pg / ml.

Examples IV-VI Using the above-described reaction technique for the preparation of substituted polypeptides, polypeptides of the following formula were prepared: For the second polypeptide, activators were observed. A PX 7812614-1 RX-LYS-Y-GLN-R 'These peptide amides were prepared on a benzhydrylamine resin by solid phase synthesis in a known manner.

Example ß Ä X _ 5: IV H SAR D-ALA NH2 IVA H SAR SAR NH2 VH D-ALA D-ALA NH2 / VA H D-ALA SAR NH2 VI 'Hr SAR 2-Me-ALA NH2 VIA H SAR SAR OH . The polypeptides prepared in Examples IV-VI retain the biological activity described herein for the active polypeptide segment. A D For identification, thin layer chromatography and electrophoresis were performed as follows.

Thin layer chromatography was performed on 20 ng Sample of gel gel (silica gel, 5 x 20 cm) using n-butanol: acetic acid: ethyl acetate: water in the ratio 1: 1: 1: 1 as eluent (Rfl) and on cellulose 606U (Eastman, 20 x 20 cm) using n-butanol: ~ pyridine: acetic acid: water in the ratio l5: 10:}: l2 as eluent (Rr2). Electrophoresis was performed with 50 g of sample on Whatman Paper No. 3 (5.7 x 55 cm) using a pyridine acetate buffer solution of pH 5.6 and a voltage of 1000 V for 1 hour. The associations ignore the cathode. Spray reagents for both thin layer chromatography and electrophoresis were Pauly and ninhydrin.

The following results were obtained (both Rf values and electrophoresis are given relative to H-ARG-LYS-ASP-VAL-TYR-OH): l 2 Electrophoresis Example ɧ_ Éf_ migratïÉÉ "" __ "FÉEHet IV 0, UN 0.68 2, 07 98% IVA 0.88 0.60 1.78 98% V 0.56 0.71 2.10 98% According to the thin layer chromatography and electrophoresis procedure described in Example I, the following results were obtained for the compound of Example VI: Rf = 0.155, Rf - immobile »Rfå = 0.265 and the electrophoresis migration is 13.1 cm towards the cathode.

Example VII Tetrapeptide resin with protected LYS and SER prepared W; The compounds of Examples I and VIA are acylated by reaction with acetic anhydride under acetylation conditions, after which the protecting groups and the resin are removed to prepare the following acylated derivatives: CH5CO-ALA-LYS-SER-GLN-OH CH3CO-SAR -LYS-SAR-GLN-OH Example VIII - Protected tetrapeptide resins prepared according to Examples I and VIA are transesterified from the resin by reaction with sodium methoxide in methyl alcohol under transesterification conditions, after which the protecting groups are removed, to prepare esterified derivatives of the following formulas: -ALA-LYS ~ SER-GLN-OCH3 H-SAR-LYS ~ SAR ~ GLN-OCH3.

Example IX The protected acetylated tetrapeptide resins prepared according to Example VII are reacted with ammonia in dimethylformamide under amidation conditions, after which the protecting groups are removed, to prepare the following peptide amides: CHBCO-ALA-LYS-SER-GLN-NH2 CH3CO-SAR- LYS-SAR-GLN-NH 2 Example Gel X-XI Using the above-described reaction technique for extending a polypeptide chain, the following polypeptides are prepared which contain the active amino acid sequence but which are substituted at the terminal amino and carboxylic acid groups R and R 'for preparation of polypeptides of the formula: In R-ALA-Lys-san-GLN-R 'xome are substituted by the amino acids indicated in the following table: Example gel 3 μl X H-GLN OH XI H-SAR OH The polypeptide derivatives prepared in Examples IV-XI retain the biological activity described above for the active polypeptide segment.

Thin layer chromatography and electrophoresis according to Example I of the compound of Examples X and XI give the following results: 10 20 BO 55 7812614-1 _22 1 2 electrophoresis RR 3 migration ÉÄÉERÉÄ _É_ fm Rf against cathode X immobile immobile 0.303 7, u cm XI immobile immobile 8 .3 Cm 0.186 Example XXVII To further illustrate the utility of the present polypeptides, this example describes a microculture assay for estimating the presence of cytotoxic lymphocytes produced upon stimulation of allogeneic antigens. The presence of cytotoxic precursors in control animals and animals injected with different concentrates of the drug was compared according to a limiting dilution analysis.

Materials and Methods Mšââ Inbreed C57 BL / 6J (females, 8 weeks) were obtained from Jackson Laboratory, Bar Harbor, Maine. 0 Inbreed DBA / 2J (males or females) were also obtained from Jackson Laboratory, Bar Harbor, Maine. A 'Msála rosfacbuffraa saiin (Pas), RPM: 16uo, fatal xaivsérum (Pos) - (lot number R776l16), N-2-hydroxyethylpiperazine-N'-2-ethanesulonic acid (HEPES) buffer was obtained from Gibo, Grand Islands; 2-Mercaptoethanol was obtained from Eastman Kodak, Rochester, N.Y.

Cells were washed with PBS and grown in RPMI containing 10% scs, 10 mm nsvs buffer and 5 x 10 5 M 2 -mercapcoecanoi.

Drug treatment The experimental animals (C57 BL / 6J) were injected (i.v. or i.p.) with different concentrations of H-SAR-Lys-SAR-cLN-NH2 <1akemea1ec; identification number GOH0) in an amount of 0.2 ml 2N hours before they were killed for the experiments.

Cell aggregate Cell suspensions from spleens from C57; BL / 6J mice or DBA / 2J mice were prepared by chopping the organ and pressing it through a wire mesh (60 gauge) with the plunger of a 5 cm 5 syringe into a falcon petri dish (falcon 3002 , 15x60 mm).

The cell suspensions. was allowed to stand at room temperature for 10 minutes so that the large pieces of tissue were allowed to settle. The cell suspensions were then transferred to a 15 ml Corning centrifuge tube (Corning 25310) and centrifuged for 10 minutes at 1500 rpm. All cell suspensions after that three minutes in a Beckmen TJ-6 centrifuge. washed at least three more times with PBS. After washing, the response cells (C57 BL / 6J) were resuspended in culture medium and counted in a Coulter counter. * DBA / 2J (stimulator cells) were resuspended in RPMI to 107 cells / ml. 30 μg of mitomycin C was added to each ml of the DBA / 2J spleen cells and the mixtures were incubated at 3700 for 30 minutes. After treatment with mitomycin C, the spleen cells were washed three times with PBS to remove any excess mitomycin C.

The DBA cells were then resuspended in the culture medium and counted in the Coulter counter.

Mixed Lymphocyte Cultures (MLC) MLC was set up in microtiter plates (Linbro Chemicals, New Haven, Conn., IS-MVC-96). Each washer contained 96 V ~ bottom recesses. The outer depressions surrounding the edge of the plate were not used for cell culture but were filled with PBS to avoid evaporation from the culture depressions. 60 samples were set up in each V-bottom tray. Typically, each tag contained three concentrations of response cells (20 of each) and one stimulator cell concentration. The response cells were usually suspended to a concentration of 7.5 x 105, 5 x 105 and 2.5 x 105 per ml and 0.1 ml was added to each well. The stimulator cell concentrations used were 10, 2.5 x 10 6, 5 x 10 6, 0.1 ml was added to each well. The same stimulator cell concentration was used for the entire plate. A control plate that contained only response cells without stimulator cells was also set up for an estimate of a background stimulation attributable to the medium. The cells were cultured for six days at 37 ° C in a humidified incubator containing 5 Z CO 2. per ml, also here Target cells The target cells used in the cytotoxic assay were a DBA mastocytoma cell line P815. The cell line was maintained by routine passage through DBA / 2J mice. 5x108 P815 cells were used for each passage and the tumor cells from the peritoneal cavity of the carriers were used four to five days after the passage. The tumor cells from the peritoneal cavity were washed three times with PBS and then labeled with CrSI at a concentration of 100 μl per 107 cells.

The labeling was done for 1 hour at 37 ° C in a humidified incubator.

The labeled target cells were then washed three times with PBS to remove any excess labeling. 10 20 25 BO 35 'Poisson. 7812614-1 Ä Cytotoxic Assay After six days of culture, 0.1 ml of medium was removed from each well without disturbing the cell pellet. Using an automatic micropipette (MLA pipette), 100 μl of target cells, containing 2.5 x 10u CrsI-labeled target cells, were then pipetted into each well, resuspending the cell pellet during the procedure (a new pipette tip must be used for each well). The microtiter plates were then centrifuged at room temperature at 1000 rpm for 7 minutes in Sorvall GLC-2B. The trays were then incubated for 4 hours at 3 ° C. 100 μl of supernatant was then removed to gamma counters (Amershen 196271). The tubes were then counted in a Beckmen gamma counter (Beckmen 310). The tubes were usually counted for 1 minute.

Determination of the presence of precursors to cytotoxic lymphocytes (CLP) The limiting dilution assay is an all or no response assay that can be described by probability distribution according to The probability of a no-answer is given by the expression of the zero order Po = e_6N where 6 = the presence of CLP and N = number of lymphocytes per well. A plot of the logarithm of the ratio of non-responsive cultures to the cell dose would thus give a straight line with a slope of -6, the occurrence or frequency of CLP.

In the example, the mean of the background release of chromium (spontaneous release) was taken from wells that contained only response cells (C57 BL / 6J) without stimulator cells. The test depths were evaluated as positive if the count value was greater than the mean spontaneous value with more than 2.07 standard deviations (P <0.05). The spontaneous lysis usually ranged from Q to 15% of the total count values present in the target cells.

According to the Poisson equation Po = e_ N then Po = e-1 = 0.57 (m0 fi SV & * rar 37% non-responding cultures), 6 = 1 / N, thus the inverse value of the number of responding cells is equivalent to -37 % non-responsive cultures CLP frequency. Typically, the number of cells per well and the corresponding value for the percentage of non-response f »entered into the computer which calculated the best regression line through these points and the number of cells per well corresponding to 57% non-response cultures were entered, the inverted value thereof being the frequency of CLP. _ 7812614-1 25 Result.

The presence of cytotoxic lymphocyte precursors for each stimulator cell concentration was plotted as a function of drug dose. For comparison, the mean and standard deviation for 20 similar samples were also calculated. The three stimulator cell concentrations used were 105 (Suboptimal stimulation), 2.5 x 105 (optimal stimulation) and 5 x 105 (over-optimal stimulation). The test drug was found to promote the production of cytotoxic lymphocyte precursors at a concentration of from about 1 pg / mouse to about 100 ng / mouse (equivalent to about 50 pg / kg to about 5 ng / kg body weight) in the presence of suboptimal stimulator cell concentrations. The experimental drug therefore acts as an immunoregulator at these concentrations, thereby increasing the cellular immune response of the treated mice.

The invention has been described here with reference to certain preferred embodiments, but insofar as obvious variations appear to the person skilled in the art, these are also covered by the invention.

Claims (3)

...,. 7s12614 ~ 1% Patent Claim A polypeptide having the ability to induce differentiation of both Th ~ 1 + T lymphocytes and Bu-1 + B lymphocytes, characterized by the following sequence: RX-LYS ~ Y-GLN ~ R 'wherein R' represents OH or NH 2, X represents sarcosyl, L-alanyl or D-alanyl, Y represents seryl, sarcosyl, 2-methyl-alanyl or D-alanyl and R represents H, H-GLN or A polypeptide according to claim 1, characterized by the following sequence: H-ALA-LYS-SER-GLN-OH and pharmaceutically acceptable salts thereof. A polypeptide according to claim 1, characterized by the following sequence: H ~ SAR ~ LYS-D ~ ALA-GLN-NH2