NO149631B - ANALOGY PROCEDURE FOR PREPARING BIOLOGICALLY ACTIVE POLYPEPTIDES - Google Patents

Description

The present invention relates to the production of biologically active polypeptides.

It is well known that many polypeptides have been isolated from different types of tissues and organs, (including blood) from different types of animals. Many of these polypeptides concern the body's immune system, for example different types of immunoglobulins, the thymus hormone thymopoietin etc. Thus, the present applicant has isolated and synthesized several of these polypeptides, as described in US patents no. 4,002,602 and 4,002,7^0 besides as described in a large number of scientific articles..

The thymus gland has been one of the least known organs in the body, and it is only in the last few decades that it has been understood that this organ is one of those that is essentially responsible for the body's immune function, this applies to both mammals and birds. It is now known, however, that the thymus gland is a complex organ with both epithelial (endocrine) and lymphoid (immunological) components, and thus forms part of the body's immune system. The thymus gland consists of an epithelial stroma derived from the so-called third gill arch and lymphocytes derived from various types of cells arising in hemopoietic tissue, Goldstein et al., The Human Thymus, Heinemann, London, 1969» The lymphocytes are differentiated within the gland and leave this as so-called mature thymus cells, so-called T cells, which circulate to the blood, lymph, spleen and lymph glands. The so-called stem cell differentiation inside the gland seems to be controlled by substances secreted differently from the epithelial cells in the thymus gland.

It has long been known that the thymus gland is connected with the body's immunity properties, and there has therefore been great interest in the substances that have been isolated from said gland. With regard to this, a number of articles have been published in recent years based on scientific investigations into the substances present in bovine thymus. Thus, the present applicant has published a number of articles relating to research in this area. Important publications in this respect can be found, for example, in The Lancet, 20o July 1968, pp. 119-122} Triangle, vol. II, no» 1, pages 7-14 1972; Annals of the New York Academy of Sciences, vol. I83, pagena 230-240 1971" and Clinical and Experimental Immunology, vol 4, no. 2, pagena 181-189, ±969} Nature, vol 247, pagena 11-14 1974; Proceedings of the National Academy of Sciences USA, vol 71, pages 1474-1478, 1974; Cell, vol. 5, pages 361-365 and 367-370, 1975j Lancet, vol. 2, pages 256-259, 1975| Proceedings of the National Academy of Sciences USA, vol 72, pages 11-15, 1975; Biochemistry, vol. 14, pages 2214-2218, 1974; Nature, vol. 255, pages 423-424, 1975.

Another type of lymphocytes. with immune function is in so-called B lymphocytes or B cells. These are formed in the bursa fabricie in birds and in a hitherto unknown organ in mammals. The T-cells and B-cells work together in many fields when it comes to the immune system. See, for example, articles in Science, 193i 319 (July 23, 1976) and Cold Spring Harbor Symposia on Quantitative Biology, vol. XLI, 5 (1977).

A non-peptide matrix has recently been isolated from pig serum by J.F. Bach et al., and identified as "facteur thymique serique" (FTS). The isolation of this substance and its structure are described in CR. Acad. Sc, Paris, vol. 283 (29 • no-vember 1976), Series D-I605 and Nature 266, 55 (3 March 1977). The structure of this non-peptide has been identified as GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN, where "GLX" represents either glutamine or pyroglutamic acid. The substance where GLX is glutamine or pyroglutarainByre has been synthesized. IN

in these articles, Bach described that said non-peptide FTS selectively differentiated T cells (and not B cells) using a so-called E-rosette test. Bach's conclusion was therefore that his substance was a thymushormoii. More detailed investigations of the activity of this non-peptide shark, however, showed that FTS differentiated both T cells and B cells, and was consequently more similar to ubiquitin in its activity than thymopoietin. Fire,

Gilmour and Goldstein, Nature, 269:597 (1977). "*

It has now been discovered that a synthesized 4-amino acid polypeptide segment from said FTS non-peptide has many of the properties described above for said non-peptide.

It is therefore an aim of the present invention to provide new polypeptide segments and polypeptides which have important biological properties, and which have the ability to induce differentiation with regard to both T-lymphocytes and B-lymphocytes, and consequently have a very wide application in the immune system both in people and animals.

The biologically active polypeptide produced according to the invention has the sequence:

where R' is OH or NH^, X is sarkosyl, L-alanyl or D-alanyl, Y is seryl, sarkosyl, 2-methylalanyl or D-alanyl,

and R is H, GLN or SAR.

The polypeptide sequence with formula I is produced according to the present invention by esterifying L-glutamine, which is protected on its amino group, to an insoluble resin polymer by covalent bonding; removing the α-amino protecting group from the L-glutamine moiety, reacting with a Y-amino acid protected on its α-amino group to link the Y-amino acid to the L-glutamine resin; removes the α-amino protecting group

from the Y-amino acid moiety, reacting with an α-amino protected L-lysine to link L-lysine to the Y-amino acid-L-glutamine resin; removes the α-amino protecting group from the L-lysine moiety, reacts with an N-R-substituted X-amino acid protected at its α-amino group to link the N-R-substituted X-amino acid to the L-lysine-Y-amino acid-L-glutamine resin, cleaves the resin from the peptide with an acid or ammonia, removing all protecting groups, to form a compound where R' is OH or NH2 respectively, provided that when the resin used is a benzhydrylamine resin polymer, then R' is necessarily NH2 regardless of the cleavage agent which is used.

The present invention thus relates to production

of new polypeptide segments and polypeptides with therapeutic value in different areas.

Whether a particular substitution results in retention of

the biological activity of polypeptides can be easily determined by testing peptides with respect to the differentiation of T-1<+> T lymphocytes and Bu-1<+> B lymphocytes in the chicken induction test described below. Compounds that are particularly active in nanogram (ng)/milliliter (ml) concentrations (approximately 1 ng/ml or less) in this sample were considered to be biologically active.

A list of natural amino acids can be found in many books, e.g. RT Morrison and R.N. Boyd, "Organic Chemistry", Allyn and Bacon, 1959, Chapter 33. In addition to the natural amino acids (which are found in proteins), there are also a number of so-called "non-natural" amino acids that are not found in proteins , but can still occur naturally as metabolic intermediates etc. These non-natural amino acids may be D-isomers corresponding to the optically active (L-form) of the natural amino acids or they may be of a completely different chemical type, such as sarcosine (N-methylglycine) or 2-methylalanine as stated mentioned above. Lists of such non-natural amino acids can also be found in many reference books.

The terminal substituents R and R' on the indicated 4-amino acid sequence must not significantly affect the biological activity of the active 4-amino acid segment, as this can be measured by the ability to induce differentiation of Th-1<+> T-lymphocytes and Bu-1<+> B lymphocytes in the above sample. As the active tetrapeptide segment is found in a longer sequence in the naturally occurring compounds isolated by Bach, it is understood that the terminal amino and carboxylic acid group is not essential for the tetrapeptide segment's biological activity, and the same applies to certain polypeptides. The present invention thus includes not only the tetrapeptide segments which are substituted with H and OH respectively, but also those which are terminally substituted with one or more other functional groups which do not significantly affect the biological activity as described here. However, it is clearly understood that the non-peptide described by Bach et al is specifically excluded from the present invention.

It appears from the above that these functional groups include such normal substitutions as occur - by acylation of the free amino group and eO-jamiderig of the free carboxylic acid group, as well as substitution with further amino acids and polypeptides. With regard to this, polypeptide segments produced according to the present invention appear to be very unusual as they show the same biological activity as the natural non-peptide of which it is itself a part. It is therefore assumed that the activity is related to the molecule's stereochemistry, i.e. to a special "bending" of the molecule. It should be emphasized here that polypeptide bonds are not rigid, but flexible, and that many polypeptides can exist as flakes, spirals, etc. As a result, the whole molecule is flexible and can bend in certain ways. In the present invention, it has been discovered that the new tetrapeptide segment presumably "bends" in a similar manner to the corresponding tetrapeptide segment in the natural non-peptide which exhibits the same biological properties. For this reason, the tetrapeptide segments can be terminally substituted with different types of functional groups as long as these substituents do not significantly affect the biological activity or affect the natural "bending"

on the molecule.

The ability of the molecule to retain its biological activity and natural bending is clearly demonstrated by the fact that tetrapeptide segments of formula I exhibit the same biological properties as the natural 9-amino acid peptide described as FTS by J.F. Bach in the above-mentioned articles. In this non-peptide, a tetrapeptide sequence produced according to the present invention can be identified within the molecule, but only in combination with other amino acids which are described in said reference. As tetrapeptide segments of formula I provide the same biological activity as said non-peptide FTS, it is clear that the amino acids and peptide chains substituted on the terminal amino acid residues of the tetrapeptide segments of formula I do not affect their biological properties.

A preferred sequence of formula I is that where X is L-alanyl, Y is L-seryl, R is hydrogen and R' is OH. This preferred embodiment can be illustrated chemically as follows:

Another preferred embodiment is where X and Y are each selected from the group consisting of sarkosyl, D-alanyl and 2-methylalanyl, and a more preferred embodiment is where X is sarkosyl, Y is sarkosyl or D-alanyl, R is hydrogen

and R' is NH 2 .

The present invention also includes the preparation of pharmaceutically acceptable salts of the polypeptides. As acids that can form salts with the polypeptides, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc. can be mentioned, as well as organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthraniic acid, cinnamic acid, naphthalenesulfonic acid or sulfanilic acid.

In this description, all the amino acid components are

in the peptide identified using commonly known abbreviations. These are listed below. The D-amino acids are indicated by placing "D" in front of the abbreviation, e.g. D-alanine is indicated as "D-ALA".

Polypeptides produced according to the present invention are 4-amino acid peptides (and their substituted derivatives) which have the same properties as those found in said 9-amino acid polypeptide FTS isolated from pig blood serum as described in the above-mentioned reference by Bach et al. The peptides of formula I are particularly characterized by their ability to induce differentiation of T precursor cells as well as B precursor cells. Some of the polypeptides are active at a concentration so low

as down to one picogram (pg)/ml in the chicken induction test described below.

The polypeptides of formula I have been found to induce the differentiation of the immunocyte progenitor cells in vitro in the same manner as the aforementioned non-peptides described by Bach. Thus, it has been found that said polypeptides induce the differentiation of both T-precursor cells, as this can be measured, as well as B-precursor cells, as this can be measured by the acquisition of differentiating antigen Bu-1. Said on

in another way, the produced polypeptides have the ability to induce differentiation of both Th-1<+> T-lymphocytes and Bu-1<+>B-lymphocytes.

It has also been found that the polypeptides of formula I

increases the ability of an in vivo production of cytotoxic lymphocytes upon stimulation of allogeneic antigens. If one thus adds the polypeptides, e.g. to rats, this promotes the production of cytotoxic lymphocyte precursors as can be measured by an in vitro test on rat spleen cells. Such a development of cytotoxic lymphocytes directly corresponds to the degree of rejection found in allogeneic grafting by a host reaction, and this discovery further supports the immunological ability and benefits that can be achieved with the help of the produced polypeptides.

To emphasize how important the differentiating biological properties of the polypeptides of formula I are, it can be noted that the function of the thymus gland with regard to immunity can be briefly indicated by the production of thymus-derived cells or lymphocytes called T cells. These T cells form a larger part of the amount of circulating small lymphocytes in the body. The T cells have immunological specificity and are directly involved in cell-controlled immune reactions (e.g. a so-called homopod reaction) as so-called effector cells. However, the T cells do not secrete so-called humoral antibodies.

These antibodies are secreted by cells (known as B cells) which are produced directly in the bone marrow independently of the thymus gland's influence. However, it has now been shown that for many types of antigens the B cells require the presence of suitable reactive T cells before they can produce antibodies. This cell-cooperative mechanism is not fully understood as of Today.

It appears from the above that the thymus gland is necessary for the development of cellular immunity and many humoral antibody reactions, and that it affects these systems by inducing, within the thymus gland, the differentiation of haemopoietic stem cells into T cells. This inductive influence is controlled by secretions from the thymus gland's epithelial cells, i.e. by thymus hormones.

In order to fully understand how the thymus gland and the cell system of lymphocytes work and how the circulation of lymphocytes in the body takes place, it should • be pointed out that said stem cells can arise in the bone marrow and reach the thymus gland with the help of the blood stream. Inside the thymus gland, the stem cells will then be differentiated into immunologically competent T cells which then migrate further into the blood stream and which, together with B cells, circulate between different types of tissue, lymph glands and the blood stream.

The body cells that secrete antibodies (B cells) also develop from hemopoietic stem cells, but this differentiation is not determined by the thymus gland. In birds, they are differentiated into an organ analogous to the thymus gland, called the bursa fabricie. No corresponding organ has been discovered in mammals, and it is assumed that these cells differentiate inside the bone marrow. They were thus designated bone marrow-derived cells or B cells. The physiological substances that govern this differentiation are completely unknown.

As mentioned above, polypeptides of formula I can be used therapeutically in the treatment of both humans and animals. As the new polypeptides have the ability to induce the differentiation of lymphopoietic stem cells that arise in the hemopoietic tissue, into both thymus-derived lymphocytes (T cells) and immuno-competent B cells that are able to participate in the body's immune reaction, products can according to the present invention have many kinds of therapeutic applications. As the compounds have the ability to perform certain anite functions in the thymus gland, they primarily have application to different thymus gland functions and different fields of immunity. A particular area of application is the treatment of DiGeorge syndrome, a condition where there is a congenital absence of the thymus gland. Injection of one of the produced polypeptides, as indicated below, will thus compensate for this deficiency. Another application is in the treatment of agammaglobulinaemia, which is due to a lack of supposed B-cell differentiating hormones in the body. Injection of one of the present polypeptides will correct this deficiency. As the present polypeptides are extremely active at low concentrations, they can be used in a general improvement of the body's immunity by increasing the therapeutic stimulation of cellular immunity and humoral immunity, and they can therefore be used in the treatment of disorders such as include chronic infection in vivo,

such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections etc. Furthermore, the produced peptides can be used in many fields where cellular or humoral immunity is included, and especially in cases where there is a deficit or a lack of immunity, so

as in the above-mentioned DiGeorge syndrome. Due to their properties, they can further be used in an in vitro induction in the development of surface antigens of T cells, in an induction with regard to the development of functional ability to achieve reactions

tion to mitogens and antigens, as well as in cell cooperation that improves the ability of B cells to produce antibodies. Furthermore, they have an in vitro application in that they induce the development of B cells, as this can be measured by the development of surface receptors for complement. The produced peptides can also be used for inhibition of uncontrolled proliferation or increase of lymphocytes due to a reaction to ubiquitin (described in US patent no. 4,002,602). An important property of the produced polypeptides is their in vivo ability to recreate cells that have the same properties as T cells, as well as their

in vivo ability to reproduce cells that have the same characteristics or abilities as B cells. They can therefore be used in treatment

ling a relative or an absolute B-cell deficiency as well as a relative or absolute T-cell deficiency, whether these deficiencies are due to deficiencies in the tissue-differentiating B cells or in the thymus gland respectively, or are due to other causes.

A very important property of the produced polypeptides is that they are very active in very low concentrations. It has thus been found that the polypeptides are generally active in concentrations of approx. 1 ng/ml, while certain potent polypeptides (H-SAR-LYS-D-ALA-GLN-NH2 and H-SAR-LYS-SAR-GLN-NH2) are active in concentrations varying from approx. 0.1 pg/ml and above The carrier may be any well-known carrier for this purpose, such as normal saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent absorptive loss to the glass at these low concentrations. The polypeptides of formula I are generally active

in an area of over approx. 1 ug/kg body weight, while some very potent polypeptides are active from approx. 1 ng/kg body weight. For the treatment of DiGeorge syndrome, the polypeptides can be administered in an amount of from approx. 1 to approx. 100 ug/kg body weight, while the potent polypeptides can be added in amounts of from 1 to 100 ng/kg body weight. The same dosage amounts can be used in the treatment of other conditions or disorders as mentioned above. Although these figures are given with regard to parenteral administration, you can of course also use oral administration in doses that vary from approx. 100 to 1000 times larger than those used for injection.

The polypeptides of formula I can be prepared by methods corresponding to those described by

Merrifield, as stated in the Journal of American

Chemical Society, 85, pp. 2149-2154, 1963. The syntheses involve a stepwise addition of protected amino acids to a growing peptide chain which is bound by covalent bonds to a solid resin particle. Using this method, reagents and by-products can be removed by filtration and the need for recrystallization of the intermediate products is eliminated. This method generally involves attaching a C-terminal amino acid in the chain to a solid polymer by means of a covalent bond and adding the subsequent amino acids one by one in a stepwise manner until the desired sequence has been assembled. Finally, the peptide is then removed from the solid support and the protective groups are then removed. This method provides a growing peptide chain fixed to a completely insoluble solid particle, so that it can be suitably filtered and washed free of reagents and by-products.

The amino acids can be attached to any suitable polymer which only needs to be easily separable from unreacted reagents. The polymer may be insoluble in the solvents used or may be soluble in certain solvents and insoluble in others. The polymer must have a stable physical form that facilitates filtration. Furthermore, it must contain a functional group to which the first protected amino acid can be firmly attached by means of a covalent bond. Various insoluble polymers which are suitable for this purpose are for example cellulose, polyvinyl alcohol, polymethacrylate and sulphonated polystyrene, but in the present synthesis a chloromethylated copolymer of styrene and divinylbenzene was usually used. You can also use polymers which are soluble in organic solvents but are insoluble in aqueous solvents. One such polymer is a polyethylene/glycol with a molecular weight of approx. 20,000, which is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide syntheses is described in F. Bayer and M. Mutter, Nature 237, 512 (1972) and the references cited therein.

The different functional groups on the amino acid

which are active, but which are not to be included in the present reactions, must be protected by means of commonly known protecting groups as these are used in polypeptide syntheses. Thus, the functional group on ^lysine must be protected by protective groups that can be easily removed after building up the desired sequence, without damaging the finished polypeptide product. In the present synthesis, fluorescamine was used to determine if the coupling was complete, which is indicated by a positive fluorescence (see Felix et al., Analyt, Biochem, 52, 377" 1973)" If incomplete coupling was indicated, then this was repeated with the same protected amino acid before possibly trying to remove the protection again. C-terminal amino acids can be attached to the polymer in a number of well-known ways. A summary of methods for attachment to halomethyl resins is given by Horiki et al., Chem Letters, pages 165-168 (1978) and Gisin, Heiv. Chim. Acta, 56, 1476 (1973) and the references indicated therein.

The present method includes, as mentioned, a first esterification of L-glutamine, which is protected on its amino groups, to the resin in an absolute alcohol containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and then dried. The protecting group on the alpha-amino group of the glutamine amino acid (eg t-BOC, ie t-butyloxycarbonyl) was then removed. The resulting coupled amino acid resin with the free amino group was then reacted with a protected L-serine, preferably alpha-t-BOX-0-benzyl-L-serine, thereby coupling the L-serine. The reactions were then repeated with protected L-lysine and L-alanine until the complete molecule had been produced. This sequence of reactions was carried out as follows:

In the above reaction sequences, R^ is a protecting group for the alpha-amino group, and R2 and R<j are protecting groups on the reactive side chains of L-serine and L-lysine, respectively, which are not affected or removed when removed to allow further reaction, In the above-mentioned intermediate pentapeptide resin, preferably R-^ is a protecting group such as t-butyloxycarbonyl, R2 stands for benzyl or substituted benzyl (e.g. k chlorobenzyl), and R^ stands for substituted benzyloxycarbonyl (fAs 2.6 -di-chlorobenzyloxycarbonyl), The resin can be any of the resins mentioned above.

After the final intermediate product is produced,

the peptide resin can be cleaved to remove the aforementioned R 1 R 2 and R 2 -protecting groups as well as the resin. The protective groups are removed in the usual way, for example by treatment with anhydrous hydrogen fluoride, and the resulting free peptide can then be recovered.

As mentioned above, it is necessary when carrying out the process to protect or block the amino groups in order to regulate the reaction and obtain the desired products. Suitable amino protecting groups which can be used include salt formation to protect strongly basic amino groups, or urethane protecting substituents such as p-methoxy benzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to use t-butyloxycarbonyl (BOC) or t-amyloxycarbonyl (AOC) to protect the alpha-amino group of the amino acids that are reacted at the carboxyl end of the molecule, as said BOC and AOC (t-amyloxycarbonyl) protecting group can be easily removed after the reaction and before the subsequent step (where such an alpha-amino group is itself reacted) by relatively weak acids (e.g. trifluoroacetic acid), and this treatment will not affect the groups used to protect other reactive side chains. It is thus understood that the alpha-amino groups can be protected by a reaction with any desired compound which will protect the amino groups in the subsequent reactions, but which can later be removed under conditions which otherwise do not affect the molecule. Examples of such compounds are organic carboxylic acid derivatives which will acylate the amino group.

Generally, any of the amino groups can be protected by reaction with a compound containing a group of the following formula:

where R^ is any group which will prevent the amino group from entering the subsequent coupling reactions, and which can be removed without destroying the molecule. Thus R^ can be a straight or branched alkyl chain which can be unsaturated, preferably with

from 1 to 10 carbon atoms, and preferably halogen- or cyano-substituted aryl, preferably with from 6 to 15 carbon atoms; cycloalkyl, preferably having from 5 to 8 carbon atoms; aralkyl, preferably having from 7 to 18 carbon atoms; alkaryl, preferably having from 7 to 18 carbon atoms; or heterocyclic groups, eg isonicotinyl. The aryl, aralkyl and alkaryl groups can further be substituted with one or more alkyl groups with from 1 to k carbon atoms. Preferred groups for R include t-butyl, t-amyl, tolyl, xylyl and benzyl. Particularly preferred amino protecting groups include benzyloxycarbonyl, substituted benzyloxycarbonyl, where the phenyl ring is substituted with one or more halogen atoms, eg chlorine or bromine; nitro; lower alkoxy, eg methoxy; lower alkyl; t-butyloxycarbonyl, t-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; bi-phenylisopropoxycarbonyl; beer. Other protecting groups which may be used include isonicotinyloxycarbonyl, phthaloyl, p-tolyl-sulfonyl, formyl and the like.

By carrying out the present method, the peptide is built up by reacting the free alpha-amino group with a compound that has protective amino groups. For the coupling itself, the compound that is attacked can be activated by its carboxyl group, so that this can then react with the free alpha-amino group on the attached peptide chain. In order to achieve activation of the carboxyl group, this can be converted into a near-reactive group such as an ester, an anhydride, azide, acid chloride, etc. Alternatively, a suitable coupling agent can be added during reaction. Suitable coupling reagents are, for example, described in Bodanszky et al. - Peptide Syntheses, Interscience, second edition 1976, chapter five, and includes compounds such as carbodiimides (e.g. dicyclocarbodiimide), carbonyldiimidizole, and the like.

It is understood that during these reactions the amino acid groups can contain both amino groups and carboxyl groups, and usually one of the groups will be included in the reaction while the other is protected. Before the coupling step, the protective group on the alpha-amino group or the terminal amino group on the attacked peptide can be removed under conditions that will not substantially protect the other protecting group, e.g. the group on the epsilon-amino group on the lysine molecule. The preferred method in this step is treatment with a weak acid, for example a reaction at room temperature with trifluoroacetic acid. Using the process described above, a tetrapeptide with the following formula is produced:

H-ALA-LYS-SER-GLN-OH II

This tetrapeptide contains one of the tetrapeptide segments that is necessary to achieve biological activity. By substituting a natural or non-natural amino acid residue for either one of the amino acids L-alanyl or L-seryl, or both of them, can be accomplished by substituting either one or both by means of suitably protected natural or non-natural amino acid in the above synthetic scheme, whereby a tetrapeptide with the following formula is obtained:

H-X-LYS-Y-GLN-OH HA

where X and Y are as defined previously. A substituted tetrapeptide of formula II, where the terminal amino acid groups can be further substituted as described above, can then be prepared by a reaction between a tetrapeptide of formula HIA or a protected peptide resin precursor with suitable reagents so that the desired derivatives are produced. Reactions of this type include acylation, esterification, amidation, etc., and are well known. Furthermore, other amino acids, i.e. amino acid groups which do not affect the biological activity of the tetrapeptide sequence, can be added to each end of the peptide chain by the same reaction sequence by means of which the tetrapeptide itself was produced. Furthermore, a substituent of either one or both of the amino acids alanine or serine can be carried out by using the desired substituent (suitably protected) instead of alanine or serine in the indicated reaction sequence whereby the unsubstituted tetrapeptide is produced.

Although the solid phase technique is usually used

which is stated by Merrifield to prepare the polypeptides of formula I, then one can of course also use known classical techniques such as this e.g. is described in M. Bodanszky 'and M.A. Ondetti, Peptide Synthesis, Interscience, 1966.

Identity and purity of the produced peptides were determined by well-known methods such as thin-layer chromatography, electrophoresis, amino acid analysis and the like.

The following examples illustrate the invention. In all examples, parts per weight unless otherwise stated.

Example I

In the preparation of the polypeptide of formula I, the following compounds were purchased commercially: Alpha-BOC-L-glutamine-o-nitrophenyl-ester Alpha-BOC-e-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-0-benzyl-L -serine

Alpha-BOC-L-Alanine.

In these reagents, BOC is equal to t-butyloxycarbonyl.

"Sequenal" quality reagents for amino acid sequence determinations, dicyclohexyl carbodiimide, fluorescamine and the resin were also purchased commercially. The resin used was a polystyrene-divinyl-benzene resin, size 200-400 mesh containing 1% divinylbenzene and 0.75 mmol chloride per grams of resin.

In the preparation of the polypeptide, 2 mmol of alpha-BOC-L-glutamine was esterified to 2 mmol of chloromethylated resin in absolute alcohol containing 1 mmol of triethylamine during 24 hours at 80°C. The resulting amino acid resin ester was filtered, washed with absolute alcohol and dried. Then, the other alpha-BOC amino acids were similarly linked to the unprotected alpha-amino group on the peptide resin in the correct order to give a polypeptide of Formula I using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, a portion of the resin was probed with fluorescamine, and if positive fluorescence was found, it was taken as evidence that the coupling was incomplete and repeated with the same protective amino acid. As a result of several coupling reactions, the intermediate tetrapeptide resin was then produced.

Said peptide resin was then cleaved and the protecting groups removed in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0°C for 60 minutes with 1.2 mmol of anisole per gram of peptide resin as absorbent. The peptide mixture was washed with anhydrous ether and extracted with aqueous acid. The extract was lyophilized, and the peptide chromatographed on "P-6 Bio-Gel."

in IN acetic acid. The resulting polypeptide was determined to be 94% pure, and had the following sequence:

H-ALA-LYS-SER-GLN-OH

For identification, thin-layer chromatography and electrophoresis were performed as follows.

Thin layer chromatography was performed with a 30 µg sample

on silica gel ("Brinkman Silica Gel" with fluorescent indicator, 20 x 20 cm, 0.1 mm thick) using the following eluents:

Rf i: n-butanol:pyridinium : acetic acid : water; 30:15:3:12

R^ 2: ethyl acetate : pyridine : acetic acid : water; 5:5:1:3

3

Rf : ethyl acetate : n-butanol : acetic acid : water; 1:1:1:1

The electrophoresis was performed on a 100 µg sample on Whatman 3 mm paper (11.5 x 56.5 cm) using a pH 5.6 pyridine-acetate buffer solution and a 1000 volt voltage for one hour.

Spraying reagents for both thin-layer chromatography and electrophoresis were Pauly and Ninhydrin.

The following results were obtained: R.<1> motionless, 2 3

Rf stationary and Rf = 0.336. The electrophoresis resulted in a migration of 9.4 cm towards the cathode.

Example II

To determine the activity and properties of the tetrapeptide prepared as described in Example I, the following chicken induction tests were performed. This sample is described in more detail in Brand et al., Science, 193 319-321 (July 23, 1976) and the references therein.

Bone marrow from newly hatched chicks was chosen as a source of inducible cells because such cells lack a

substantial number of Bu-1<+> or Th-l<+> cells. Pooled cells from the thigh and from the tarsus of five newly hatched chickens of the breed SC (Hy-Line) were fractionated by ultracentrifugation on a five-layer discontinuous bovine serum albumin (BSA) gradient. The cells from the two lightest layers were pooled, washed, and suspended for incubation at a concentration of 5 x 10^ cells per milliliter with an appropriate concentration of the test polypeptide in RPMI 1630 medium supplemented with 15 mmol of hepes, 5 percent gamma-globulin-free fetal calf — serum, deoxyribonuclease (1 k to 18 units/ml), heparin (5 units/ml), penicillin (100 units/ml) and streptomycin (100 M-g/ml). The controls were incubated with BSA (in |ig/

ml) or with medium alone. After incubation, the cells were tested in the cytotoxicity test using chicken C1 and guinea fowl 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, where a and b are the percentage of live cells in the complement control and the sample preparation respectively. The percentage of cells induced was obtained by subtracting the means in the control incubations without inducer (usually 1 to 3 percent) from the means obtained in the sample inductions.

The test polypeptide's specificity of action and its similarity to ubiquitin was shown by inhibition of the induction of Bu-1<+ >B cells and Th-1<+> T cells when the test polypeptide was added to ubiquitin in a concentration of 100 (ug/ml. This high dose of ubiquitin inactivates the ubiquitin receptors and thus prevents the induction of cells by any agent that acts through these receptors.

As a result of this test, it was discovered that the tetrapeptide from example 1 has similar biological activity to ubiquitin in that it induces the differentiation of both Th-1<+> Tog Bu-1 + B lymphocytes in ng/ml concentrations.

Example III

A. The sample from Example II was repeated using as a sample polypeptide one of the following: H-GLN-ALA—LYS-SER-GLN-GLY—GLY—SER-ASN-OH H-GLN-ALA-LYS-SER-GLN -OH

H-SAR-ALA-LYS-SER-GLN-OH

In each case, a biological activity corresponding to that found in ubiquitin was obtained.

B. The sample of Example II was repeated using a sample polypeptide of one of the following:

H-SAR-LYS—D-ALA-GLN—NH2

H-SAR-LYS-SAR-GLN-NH2

H-D-ALA-LYS-D-ALA-GLN-NH2

In each case, a biological activity corresponding to that found in ubiquitin was again found. For the first of these polypeptides, it was found that this activity was in the concentration range from approx. 1 pg/ml to approx. 100 pg/ml. For the second polypeptide, activity was observed at a concentration as low as 0.1 pg/ml.

Examples IV - VI

By using the reaction technique described above for the production of substituted polypeptides, polypeptides with the following formula were produced:

R-X-LYS-Y-GLN-R<1>

These peptidamides were prepared on a benzhydrylamine resin by the so-called solid phase synthesis technique.

The polypeptides prepared as set forth in Examples IV-VI retained the biological activity of the active polypeptide segment.

For identification, thin-layer chromatography and electrophoresis were carried out in the following manner.

Thin layer chromatography was performed on a 20 µg sample

on silicon dioxygel (Kieselgel, 5 x 20 cm) using as eluent n-butanol:acetic acid:ethyl acetate:water in a quantity ratio" of 1:1:1:1 (Rf<1>) and on cellulose 6o6k (Eastman, 20 x 20 cm) using as eluent n-butanol:pyridine:acetic acid:water in a ratio of 15:10:3:12 (Rf ).

Electrophoresis was performed on 50 µg of sample on Whatman No. 3 paper (5.7 x 55 cm) using a pH 5-6 pyridine-acetate buffer solution and a voltage of 1000 volts for one hour. The compounds migrate towards the cathode.

Spraying reagents for both the thin-layer chromatography and the electrophoresis were Pauly and Ninhydrin.

The following results were obtained (both the Rf. values and electrophoresis. are indicated in relation to H-ARG-LYS-ASP-VAL-TYR-OH):

Examples VII and VIII

By using the reaction technique described here for an extension of the polypeptide chain, the following polypeptides were prepared which all contain the active araino acid sequence, but which are substituted on the terminal amino and carboxylic groups R and R' so that one obtains a polypeptide of the following formula:

R-ALA-LYS-SER-GLN-R'

which were substituted with the amino acids indicated in the table below.

Polypeptide derivatives prepared as indicated in Examples IV-VIII retain the biological activity possessed by the active polypeptide segment.

Using thin layer chromatography and electrophoresis as described in Example I for the compounds from Examples VII and VIII, the following results were obtained:

Example IX

To further illustrate the utility of the present polypeptides, this example describes a microculture assay to determine the frequency of cytotoxic lymphocytes produced upon stimulation with allogeneic antigens. The frequency of cytotoxic precursors between the control animals and animals injected with different concentrations of the tested compound was compared by a so-called limiting dilution test.

Materials and methods

Mouse

Inbreed C57 BL/6J (females, 8 weeks) were obtained from Jackson Laboratory, Bar Harbor, Maine.

Inbreed DBA/2J (males or females) were also obtained from Jackson Laboratory, Bar Harbor, Maine.

Media

Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS) - (lot number R776116), N-2-hydroxy-ethylpiperazine-N<1->2-ethanesulfonic acid (HEPES) buffer;

2-mercaptoethanol. Cells were washed with PBS and cultured in RPMI containing 10% FCS, 10 mmol HEPES buffer and 5x10 mol 2-mercaptoethanol.

Medical treatment

The test animals (C57 BL/6J) were injected (i.v. or i.p.) with different concentrations of H-SAR-LYS-SAR-GLN-NH2 (the drug; identification no. G040) in a 0.2 milliliter volume 24 hours before they were necropsied for the experiments.

Cell preparations

Cell suspensions from the spleens of C57 BL/6J mice or DBA/2J mice were prepared by grinding the organ and pressing it through a fine mesh (60 gauge) using the plunger of a 5 cubic centimeter syringe into a Petri dish (Falcon 3002, 15x60 mm). The cell suspensions were left at room temperature for 10 minutes so that the larger clumps of tissue became centimeter-two. The cell suspensions were then transferred to 15 milliliter corning centrifuge tubes (Corning 25310) and spun for 10 minutes at 1500 rpm in a Beckmen TJ-6 centrifuge . All cell suspensions were washed at least three times with PBS. After the third wash, the remaining (C57 BL/6j) cells were resuspended in culture medium and counted in a Coulter counter. DBA/2J (stimulator cells) were resuspended in RPMI to 10 cells per milliliter. 30 µg of mitomycin C was added to each milliliter of DBA/2J spleen cell, and the mixtures were incubated at 37°C for 30 minutes. After the mitomycin C treatment, 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 microtitre dishes (Linbro Chemicals, New Haven, Connecticut, IS-MVC-96). Each dish contained 96-V bottom holes. The outer holes surrounding the edge of the plate were not used for cell cultures but were filled with PBS to avoid evaporation from the culture holes. 60 samples were used in each V-bottom dish. Typically, each dish contained three cell concentrations (20 replicates of each) and one stimulator cell concentration. The reacting cells were usually suspended to concentrations of 7.5 x 10 - 5 , 5 x 10 -^ and 2.5 x 10 - - per milliliters and 0.1 milliliters were added to each well. The stimulator cell concentrations were 10 , 2.5x10 , 5x10° per milliliters, and here too 0.1 milliliters was added to each hole. The same stimulator concentration was used throughout the plate.0 The control plate contained only responding cells without stimulator cells, and this was done to assess the background stimulation that washed the medium. The cells were cultured. for six days at 37°C in a humidified incubator containing 5$> 002*

Target cells

The target cells used in this cytotoxic test were a DBA mastocytoma cell line P815. The cell line was maintained by routine passage through DBA/2J mice. 5x10<g>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

51

were washed three times with PBS and labeled with Cr at a concentration of 100 myci per 10<?> cells. The labeling was carried out for one hour at 37°C in a humidified incubator. The labeled target cells were then washed three times with PBS to remove any excess labeling reagent.

Cvtotoxic test

After six days of culture, 0.1 milliliter of the medium was removed from each hole without disturbing the cell clump. By then using an automatic micropipette (MLA pipette), 100 μl of the target cells containing 2.5*10^ Cr-'1 labeled target cells were pipetted into each hole, after which the cells were resuspended (a new pipette was used in each hole)» The microtiter plates were then spun at room temperature at 1000 rpm. for seven minutes in a Sorvall GLC-2B centrifuge. The dishes were then incubated for four hours at 37°C. 100 microliters of the overlying liquid was then removed and placed in Gamma measuring tubes (Amershen 196271). The tubes were then counted in a Beckmen Gamma counter (Beckmen 310). The tubes were usually counted for one minute.

Determination of the frequencies of cytotoxic lymphocyte precursor cells (CLP)

The limiting dilution analysis is a one or no reaction sample described by the Poisson probability distribution. The probability of a non-reaction is given by the zero-order term Po = e**Gamma N where Gamma = the frequency of CLP and N = the number of lymphocytes per hole. Thus, a plot of the logarithm of the amount of non-reacting cultures in relation to the cell dose should give a straight line with a slope of - Gamma, ie the frequency of CLP.

In the examples, means were calculated of the chromium release that was washed into the medium or the background (spontaneous release) from 20 cells that only contained reacting cells (C57 BL/6J) without stimulator cells. Sample wells were considered positive if their counts were greater than the mean spontaneous value by more than 2.07 standard deviations (P less than 0.05). The spontaneous resolution was usually from 9-15$ of the total counts included in the counts of the target cells. According to the Poisson equation Pore^^^, where Po=e"<*1>= 0.37 (equivalent to 37$ non-reacting cultures), delta = l/N, so that the resi-proced value of the reacting cell numbers, which corresponds to 37$ non-reacting cultures is the CLP frequency.Typically, the number of cells per well and their corresponding percent non-reacting values were entered into a calculator which calculated the best regression line through these points and the number of cells per .holes which corresponded to 37$ non-reacting cultures, and the representative value of this value is the frequency of said CLP.

Results

The frequencies of cytotoxic lymphocyte progenitor cells for each stimulator cell concentration were plotted as a function of drug dose. Means and standard deviations for twenty parallels were also plotted as a comparison. The three stimulant concentrations used were 10 2 (suboptimal stimulation), 2.5 x 10 2 (optimal stimulation) and 5 x 10 -* (superoptimal stimulation). It was found that the tested compounds promoted the production of cytotoxic lymphocyte precursor cells at concentrations from approx. 1 pgram/pr. mice to approx. 100 ng/mouse (equivalent to approx. 50 pgram/kg to approx. 5 pg per kg body weight)

in the presence of suboptimal stimulator cell concentrations. The added compound therefore acts as an immuno-regulator at these concentrations and increases the cell immunity reaction in the treated mice.

Claims (1)

Analogous method for the production of the biologically active polypeptide with the sequence: R-X-LYS-Y-GLN-R' (I) where R' is Oli or NH^, X is sarkosyl, L-alanyl or D-alanyl, Y is seryl, sarkosyl, 2-methylalanyl or D-alanyl, and R is H, GLN or SAR, characterized in that L-glutamine, which is protected on its amino group, is esterified to an insoluble resin polymer by covalent bonding; removing the α-amino protecting group from the L-glutamine moiety, reacting with a Y-amino acid protected on its α-amino group to link the Y-amino acid to the L-glutamine resin; removing the α-amino protecting group from the Y-amino acid moiety, reacting with an α-amino protected L-lysine to link L-lysine to the Y-amino acid-L-glutamine resin; removes the α-amino protecting group from the L-lysine moiety, reacts with an N-R-substituted X-amino acid protected at its α-amino group to link the N-R-substituted X-amino acid to the L-lysine-Y-amino acid-L-glutamine resin, cleaves the resin from the peptide with an acid or ammonia, removing all protecting groups, to form a compound where R' is OH or NH^ respectively, provided that when the resin used is a benzhydrylamine resin polymer, then R' is necessarily NH 2 whichever cleavage agent used.