JPS6327360B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to novel polypeptides, particularly those having the following sequence: R-X-LYS-Y-GLN-R' where X is ALA, D-ALA or SAR; Y is SER , D-ALA, SAR or 2-
Me-ALA; R is H, GLN or SAR
and R' is OH or _NH2 . It is well known that many polypeptides have been isolated from various tissues and organs of animals, including blood. Many of these polypeptides, such as various immunoglobulins, the thymic hormone thymopoietin, etc., are involved in immune function throughout the body. In fact, Applicants have isolated and synthesized some of these polypeptides as described in US Pat. Nos. 4,002,602 and 4,002,740 and in several chemical papers. Until about the past decade, little was known about the thymus, but it is now understood that the thymus is one of the major organs essential for immune function in mammals and birds. Until recently, little was known about the function of the thymus, despite intense interest in its possible function and early speculation and experiments. However, as is now recognized, the thymus is a complex organ with both epithelial (endocrine) and lymphoid (immunological) components, and as such it is involved in the body's immune function. The thymus is composed of epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells derived from hemopoietin tissue.
Goldstein, et al., The Human Thymus ,
Heinemann, London, 1969. Lymphocytes are mature thymus-derived cells within the thymus and lobes, so-called T
These T cells circulate in the blood, lymph, spleen, and lymph nodes. Induction of stem cell differentiation within the thymus appears to be mediated by secretion of thymic epithelial cells. The thymus has been known for some time to be associated with the immune properties of the body, and therefore great interest has been shown in substances isolated from the thymus. In this regard, a relatively large number of papers have recently been published based on scientific studies on the substances present in the thymus of cows. In fact, the applicant has published numerous articles on research in this field. Relevant publications can be found, for example: The Lancet , July 20, 1968, pp. 119−
122; Triangle , Vol., No.1, pp.7−14,
1972; Annals of the New York Academy of
Sciences, Vol.183, pp.230−240, 1971; and
Clinical and Experimental Immunology;
Vol.4, No.2, pp.181-189, 1969; Nature ,
Vol.247, pp.11−14, 1974; Proceedings of the
National Academy of Sciences USA,
Vol.71, pp.1474−1478, 1974; Cell , Vol.5,
pp.361−365 and 367−370, 1975; Lancet ,
VoL.2, pp.256−259, 1975,; Proceedings of
the National Academy of Sciences USA,
Vol.71, pp.1474−1478, 1974; Cell , Vol.5,
pp.361−365 and 367−370, 1975; Lancet ,
Vol.2, pp.256−259, 1975; Proceedings of the
National Academy of Sciences USA,
Vol.72, pp.11−15, 1975; Biochemistry ,
Vol.14, pp.2214−2218, 1974; Nature ,
Vol.225, pp.423−424, 1975. The second class of lymphocytes with immune function are B lymphocytes or B cells. These are the Bertha of Fabricius in birds.
Bursa of Fabricius) and by organs yet to be identified in mammals. T cells and B cells cooperate in many aspects of immunity. References, e.g. papers by the applicant,
Science, 193 , 319 (July23, 1976) and Cold
Spring Harbor Symposia on Quantitative
Biology, Vol.XLI, 5 (1977). Nonapeptide substances were recently extracted from pig serum by JF
Bach, et al. and “facteur
thymique serique” (FTS). The isolation of this substance and its structure were published in CRAcad.Sc.Paris ,
t, 283 (November 29, 1976), Series D-1605
and Nature 266 , 55 (March 3, 1977). The structure of this nonapeptide is GLX−
ALA−LYS−SER−GLN−GLY−GLY−SER
-ASN, where "GLX" stands for glutamine or pyroglutamic acid. GLX
A substance in which is glutamine or pyroglutamic acid has been synthesized. In these papers, Bach discloses that his nonapeptide FTS selectively differentiated T cells (but not B cells) by use of the E-Rosette assay. Bach therefore concluded that his substance was a thymic hormone. Recently, a more complete study of the activity of this nonapeptide according to the present application revealed that FTS differentiates both T and B cells and therefore its activity is more similar to ubiquitin than thymopoeitin. It was revealed that there was. Brand, Gilmour and
Goldstein, Nature , 269 :597 (1977). It has now been found that the synthesized 4-amino acid polypeptide segment of this FTS nonapeptide has many of the properties of nonapeptides discussed in the above publications. One objective of the present invention is therefore to provide novel polypeptides of biological importance. Another object of the invention is to provide novel polypeptides that have the ability to induce differentiation of both T-lymphocytes and B-lymphocytes and are therefore highly useful in the human and animal immune system. That's true. The subject polypeptides can be described by the following general formula: . R-X-LYS-Y-GLN-R' where X and Y are as previously described and R is H, GLN or SAR; and
R' is OH or NH ₂ and the peptide segment does not substantially affect the biological activity of the active amino acid segment, as described above. Since the active tetrasegment is contained within a long sequence in the naturally occurring material isolated by Bach, the terminal amino group and the terminal carboxylic acid group, as in the case of some polypeptides, It is to be understood that there is no requirement for biological activity of the tetrapeptide segment. .
Therefore, the scope of the present invention is H and
It is believed to encompass not only tetrapeptide segments substituted with OH, but also those terminally substituted with one or more other functional groups that do not affect the biological activity disclosed herein. However, it is clearly understood that the nonapeptides described by Bach et al. are specifically excluded from the scope of the present invention. From this description, it will be understood that these functional groups include common substitutions such as acylation on free amino groups and amidation on free carboxylic acid groups, as well as additional amino acid and polypeptide substitutions. In these respects, the polypeptide segments of the invention appear to be highly unusual since they exhibit the same biological activity as the natural nonapeptide of which the active tetrapeptide segment forms a part.
It is therefore believed that this requirement of the molecule arises from its stereochemistry, ie the special "fold" of the molecule. In this regard, it should be understood that polypeptide bonds are flexible rather than rigid, and that polypeptides can exist as sheets, helices, etc. After all, all molecules are flexible and will fold in some way. In the present invention, it has been discovered that the novel tetrapeptide segments will likely "fold" in a manner similar to the corresponding tetrasegments in natural nonapeptides, exhibiting the same biological properties.
For this reason, tetrapeptide segments can be terminally substituted with various functional groups, as long as the substituents do not substantially affect biological activity or interfere with the natural "folding" of the molecule. be able to. The biological activity and ability of this molecule to retain its natural folding have been demonstrated in the above-disclosed paper by JFBach.
This is clearly exemplified by the fact that it exhibits the same biological activity as the natural 9-amino acid peptide disclosed as FTS. In this nonapeptide, one tetrapeptide sequence of the invention is intramolecular, but only visible in combination with the other amino acids described here. Since the tetrapeptide segment of the present invention provides the same biological activity as the nonapeptide FTS, it is clear that the amino acids on the terminal amino acid residues of the tetrapeptide segment and the peptide chain do not affect its biological properties. . Therefore, R and R' in the formula represent the following substituents. R R' Hydrogen OH GLN NH ₂ SAR One preferred embodiment of the invention provides that X is L-alanyl, Y is L-seryl, R is hydrogen, and R' is
It's time to be OH. This preferred embodiment can be chemically symbolized as: A second preferred embodiment is that X and Y are each sarcosyl, D-alanyl, and Y is further 2-
selected from the group consisting of methylalanyl; in a most preferred embodiment, X is sarkosyl, Y
is sarcosyl or D-alanyl, R is hydrogen, and R' is _NH2 . Also included within the scope of the invention are pharmaceutically acceptable salts of the polypeptides. As acids capable of forming salts with polypeptides, the following acids may be mentioned by way of example: inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc., and organic acids. 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, anthranilic acid, citric acid, naphthalenesulfonic acid or sulfanilic acid. Throughout this invention, the amino acid components of peptides are conveniently designated by abbreviations. These abbreviations are as follows. D-amino acids are designated by placing a "D" in front of the abbreviation, eg, D-alanine is represented by "D-ALA." Amino acid abbreviations L-alanine ALA L-aspartic acid ASP L-asparagine ASN L-serine SER L-glutamic acid GLU L-glutamine GLN L-leucine LEU L-lysine LYS L-threonine THR glycine GLY L-valine VAL Sarcosine SAR 2 -Methylalanine 2-Me-ALA The polypeptides of the present invention are 4-amino acid peptides (and substituted derivatives thereof),
These were found to exhibit properties similar to the 9-amino acid polypeptide FTS isolated from bovine blood as disclosed in the Bach et al article referenced above. The peptides of the invention are particularly characterized in their ability to induce differentiation of T-precursor cells as well as B-precursor cells. Certain of the subject polypeptides are active at concentrations as low as 1 picogram (pg)/ml in the chicken challenge assay described below. It has been found that the polypeptides of the present invention induce differentiation of immunocyte precursor cells in vitro in the same manner as the nonapeptides disclosed by Bach. Thus, the polypeptides of the invention can be used to increase T-precursor cells, as measured by the acquisition of the thymic differentiation antigen Th-1, as well as B-precursor cells, as measured by the acquisition of the differentiation antigen Bu-1. It was found to induce both differentiation of cells. In other words, the subject polypeptide is
It has the ability to induce differentiation of both Th-1 ⁺ T- and Bu-1 ⁺ B-lymphocytes. The subject polypeptides have also been found to increase the ability to produce cytotoxic lymphocytes in vivo when stimulated with allograft antigens. Thus, the subject polypeptides, when administered to, for example, rats, promote the production of cytotoxic lymphocyte precursors as measured by in vitro assays of rat splenocytes. The above findings further support the immunological utility of the subject polypeptides, since the development of cytotoxic lymphocytes directly corresponds to the extent of graft rejection in allograft versus host reactions. To provide an understanding of the importance of the differentiated biological properties of the polypeptides of the invention, we state that the function of the thymus involved in immunity is the production of thymus-derived cells or lymphocytes, broadly referred to as T cells. It should be noted that it is possible to T cells form a large proportion of the pool of recirculating small lymphocytes. T cells have immunological specificity and are involved in cell-mediated immune responses (e.g., allograft responses).
directly included as responder cells. However, T cells do not secrete humoral antibodies. These antibodies are not secreted by cells derived directly from the bone marrow (called B cells) independent of thymic influence. However, for many antigens, B cells require the presence of appropriate reactive T cells before they can produce antibodies. The mechanism of this cell cooperation is also not completely understood. From this explanation, in an operational context, the thymus is required for the development of cellular immunity, and many humoral antibodies respond and activate these systems by inducing differentiation of hemopoietin stem cells into T cells within the thymus. It can be said that it has an influence on the The effect of this induction is on the secretion of thymic epithelial cells, i.e.
Mediated by thymic hormones. Furthermore, in order to understand the operation of the thymus gland and the cell lineage of lymphocytes, it should be pointed out that stem cells arise in the bone marrow and reach the thymus gland by the blood flow.
Within the thymus, stem cells become differentiated into immunologically competent T cells, which migrate into the bloodstream and circulate with B cells between tissues, lymph, and bloodstream. Cells of the body that secrete antibodies (B cells) are also expressed from hemopoietin stem cells, but their differentiation is not determined by the thymus. In birds, they contain an organ similar to the thymus, the so-called Bursa of Fabricius.
Fabricius). In mammals,
No equivalent organ has been discovered, and these cells are thought to differentiate within the bone marrow. They are therefore termed myeloid derived cells or B cells. The physiological substances responsible for this differentiation remain completely unknown. As pointed out above, the polypeptides of the invention are useful therapeutically in human and animal treatments. The novel polypeptide has the ability to induce the differentiation of lymphoid stem cells derived from hemopoietin tissues into both thymus-derived lymphocytes (T cells) and immunoadaptive B cells that can engage in the body's immune response. Therefore, the products of the present invention are believed to have numerous therapeutic uses. First, this compound has the ability to perform some of the functions exhibited by the thymus and thus has applications in various thymic functions and immunological fields. The first field of application is DiGeorge Syndrome,
In other words, it is a treatment for a condition that is the congenital absence of the thymus gland. As described further below, injection of one of the subject polypeptides will overcome this deficiency. Another application is in agammaglobulinemia, which is a deficiency of the body's putative B cell differentiation hormone. Injection of one of the subject polypeptides will overcome this deficiency. Since the subject polypeptides are highly active at low concentrations,
It is useful for promoting the body's collective immunity in increasing the therapeutic stimulation of cellular and humoral immunity, thereby preventing diseases involving chronic infections in vivo, such as fungal or mycoplasmal infections, tuberculosis, leprosy. , useful in the treatment of acute and chronic viral infections, etc. Additionally, the subject peptides are believed to be useful in areas where cellular or humoral immunity is impetigo, particularly where there is a deficiency in immunity such as the aforementioned DiGeorge Syndrome. Additionally, polypeptides have the ability to induce surface antigen expression on T cells, to respond to mitogens and antigens, and to achieve mitotic potential, which enhances the ability of B cells to produce antibodies. is useful in vitro in inducing the expression of They are useful in vitro in inducing B cell expression as measured by the expression of surface receptors for complement. The subject peptides also inhibit the uncontrolled proliferation of lymphocytes that are responsive to ubikinin (as described in Applicant's U.S. Patent No.
4002602). An important property of the subject polypeptides is their in vivo ability to restore cells with T-cell characteristics, and also their in vivo ability to restore cells with B-cell characteristics. Therefore they are relative or absolute B
useful in the treatment of cell deficiencies as well as relative or absolute T cell deficiencies, and whether these deficiencies are due to a deficiency of B cells or thymus-differentiating tissue, respectively, or to other causes. is irrelevant. Other important properties of the polypeptides of the invention are:
that they are active at very low concentrations. That is, polypeptides generally have approximately 1n
g/ml, but one highly potent polypeptide (H-SAR-LYS-D-ALA
-GLN- _NH2 and H-SAR-LYS-SAR-
GLN- _NH2 ) is active at concentrations ranging from about 0.1 pg/ml. The carrier can be any of the well-known carriers including standard saline solution, preferably a protein diluent such as bovine serum albumin that prevents adsorption loss to glassware at these low concentrations. . Although the polypeptides of the invention are generally active in the range of about 1 μg/Kg body weight or higher, certain highly potent polypeptides are
Active from 1 ng/Kg body weight. DiGeorge
For the treatment of syndrome, the polypeptide is approximately 1
It can be administered at a rate of ~100 μg/Kg body weight, but highly potent polypeptides can be administered at a rate of about 1 to about 100 μg/Kg body weight.
It can be administered at the rate of g/Kg body weight. Generally, the same range of dosages can be used for other conditions or diseases mentioned. Although the above considerations were given regarding parenteral administration,
Oral administration is also possible in a dosage range that is generally about 100 to 1000 times higher than injection. The polypeptide of the present invention can be used in the Journal of
American Chemical Society, 85, pp2149−
2154, was produced using ideas similar to those reported by Merrifield in 1963. The synthesis involved the stepwise addition of protected amino acids to a growing peptide chain covalently attached to solid resin particles. This method removed reagents and by-products by filtration and eliminated recrystallization of intermediates. The general idea of this method relies on the covalent attachment of the C-terminal amino acid of the chain into a solid polymer and the addition of one successive amino acid at a time in a stepwise manner until the desired sequence is composed. do. Finally, the peptide is removed from the solid support and
Then the protecting group is removed. Because this method provides the growing peptide chain bound into completely solid particles, it is a convenient form for washing away reagents and by-products. Amino acids can be attached to any suitable polymer, which simply must be easily separated from unreacted reagents. The polymer can be insoluble in the solvent used, or it can be soluble in some solvents and insoluble in others. The polymer should have a stable physical form that makes it easy to pass. It must contain a functional group to which the first protected amino acid can be tightly bound covalently. Examples of various polymers suitable for this purpose are cellulose, polyvinyl alcohol, polymethacrylates and sulfonated polystyrene, although chloromethylated copolymers of styrene and divinylbenzene were generally used in the synthesis of the present invention. It is also possible to use polymers that are soluble in organic solvents but insoluble in aqueous solvents. One such polymer is soluble in methylene chloride but insoluble in water, with a molecular weight of about
20000 polyethylene/glycol.
The use of this polymer in peptide synthesis is F.
Bayer and M. Mutter, Nature 237 , 512 (1972)
and the references contained therein. Various functional groups on the amino acids that were active but not participating in the reaction were protected during the reaction with common protecting groups used in the polypeptide art. The functional group on the lysine was thus protected with a protecting group that could be removed upon completion of the sequence without adversely affecting the final polypeptide product. In the synthesis, fluorescent amines were used to determine whether the bond was completed by positive fluorescent indication (see Felix, et al., Analyt, Biochem. , 52;
377, 1973). If complete conjugation was not indicated, conjugation was repeated using the same protected amino acid before deprotection. The C-terminal amino acid can be attached to the polymer in a variety of well known ways. A summary of methods for coupling to halomethyl resins can be found in Horiki, et al., Chem Letters.
pp165−168 (1978) and Gisin, Helv.Chim.
Acta, 56 , 1476 (1973) and the references cited therein. The general method is to protect L-
It involved first esterifying glutamine to a resin in an amine-containing absolute alcohol. The bound amino acid resin was then filtered, washed with alcohol and water, and dried. The protecting group (eg, t-BOC, ie, t-butyloxycarbonyl) on the α-amino group of the glutamine amino acid is then removed. The resulting bound amino acid resin with free amino groups is then reacted with protected L-serine, preferably alpha-t-BOC-O-benzyl-L-serine, to couple the L-serine. The reaction is then continued with the protected L-
Repeated with lysine and L-alanine. The order of the reactions performed was as follows: [Table] ↓
[Table] ↓
H−Ala−Lys−Ser−Gln−OH
In the above reaction sequence, R ₁ is the protecting group for the α-amino group, and R ₂ and R ₃ are the protecting groups on the reactive side chains of L-serine and L-lysine, respectively, which are R When ₁ is removed and further reactions are carried out, it is unaffected or not removed. Preferably, in the above intermediate pentapeptide resin, R ₁ is a protecting group, such as t
-butyloxycarbonyl, R ₂ is benzyl or substituted benzyl (e.g. 4-
chlorobenzyl) and R ₃ is substituted benzyloxycarbonyl (e.g. 2,
6-dichlorobenzyloxycarbonyl). This resin is any of the resins mentioned above as it is useful in this method. After making the final intermediate, the peptide resin is cleaved to remove the R ₁ , R ₂ and R ₃ protecting groups thereon and the resin. The protecting groups are removed by conventional means, such as treatment with anhydrous hydrogen fluoride, and the resulting free peptide is then recovered. As pointed out above, in carrying out this process it is necessary to protect or block the amino groups in order to control the reaction and obtain the desired product. Examples of suitable amino protecting groups that can be usefully used include salt formation to protect strongly basic amino groups, or urethane protecting substituents such as p-methoxybenzyloxycarbonyl and t-butyloxycarbonyl. be. It is preferable to use t-butyloxycarbonyl (BOC) or t-amyloxycarbonyl (AOC) to protect the α-amino group in the amino group that undergoes the reaction at the carboxyl end of the molecule. This is because the BOC and AOC (t-amyloxycarbonyl) protecting groups are treated with relatively mild acids (e.g., trifluoroacetic acid) following such reactions and before subsequent steps (such amino groups themselves carry out the reaction). ) and this treatment does not otherwise affect the groups used to protect other reactive side chains. in this way,
The α-amino group may be protected by reaction with any substance that protects the amino group for subsequent reactions, but which can subsequently be removed under conditions that do not otherwise affect the molecule. Examples of such substances are organic carboxylic acid derivatives that acylate amino groups. Generally, any amino group is Protection can be achieved by reaction with a compound containing a group in which R ₄ is any group that prevents the amino group from participating in subsequent coupling reactions and that can be removed without decomposing the molecule. R ₄ can thus be unsaturated, preferably having 1 to 10 carbon atoms and preferably halo- or cyano-substituted straight-chain or branched alkyl; aryl,
Preferably aryl of 6 to 15 carbon atoms; cycloalkyl, preferably cycloalkyl of 5 to 8 carbon atoms; alkaryl, preferably alkaryl of 7 to 18 carbon atoms; or a heterocyclic group, such as isonicotinyl. Aryl, aralkyl and alkaryl moieties can also be further substituted with one or more alkyl groups of 1 to about 4 carbon atoms. Examples of preferred groups for R are t
-butyl, t-amyl, tolyl, xylyl and benzyl. Examples of specific amino protecting groups that are highly preferred are: benzyloxycarbonyl; substituted benzyloxycarbonyl, where the phenyl ring is substituted with one or more halogens, such as Cl or Br. ; nitro; lower alkoxy, such as methoxy; lower alkyl; t-butyloxycarbonyl, t-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; biphenylisopropoxycarbonyl and the like. Examples of other protecting groups that can be used are isonicotinyloxycarbonyl, phthaloyl, p
-Tolylsulfonyl, formyl, etc. In practicing the general method of the invention, peptides are created by the reaction of a free α-amino group with a compound having a protected amino group. For reaction or conjugation, the attacked compound is activated at its carboxyl group so that the carboxyl group can then react with a free amino group on the attached peptide chain. In order to achieve activation,
The carboxyl group can be converted to any reactive group, such as ester, anhydride, azide, acid chloride, and the like. Alternatively, a suitable binding reagent can be added during the reaction. Suitable coupling reactions include carbodiimides (e.g., dicyclocarbodiimide), carbonyldiimidizole, etc.
For example, Bodanszky, et al.−Peptide
Synthesis, Interscience, 2nd edition, 1976, section 5. During these reactions, the amino acid moiety contains both an amino group and a carboxyl group, and usually one group participates in the reaction while the other is protected. Prior to the coupling step, the protecting group on the alpha or terminal amino group of the attacked peptide is removed under conditions that do not substantially affect other protecting groups, such as the group on the epsilon-amino of the lysine molecule. A preferred method of carrying out this step is mild acidolysis, such as by reaction with trifluoroacetic acid at room temperature. As can be seen, the above sequence of steps results in a tetrapeptide of the following formula: . H-ALA-LYS-SER-GLN-OH This tetrapeptide contains one tetrapeptide segment of the invention required for biological activity. Substitution of natural or non-natural amino acid residues for one or both of L-alanyl and L-seryl allows one or both of alanine and serine to be replaced with appropriately protected natural or non-natural amino acids in the synthesis diagram above. , thus producing a tetrapeptide of the formula: AH-X-LYS-Y-GLN-OH where X and Y are as defined above. A substituted tetrapeptide of the formula, in which the terminal amino acid group can be further substituted as described above, is then prepared by reacting the tetrapeptide of formula (A) or a protected peptide resin precursor with a suitable reagent to form the desired derivative. It can be manufactured by manufacturing. Reactions of this type, such as acylation, esterification, amidation, etc., are of course well known in the art. Additionally, other amino acids, ie, amino acid groups that do not affect the biological activity of the tetrapeptide sequence, can be added to either end of the peptide chain by the same reaction sequence in which the tetrapeptide itself was synthesized. Furthermore, substitution of one or both of the alanine or serine moieties may be accomplished by substituting the desired substituent (suitably protected) for the alanine or serine in the preceding reaction sequence that synthesized the unsubstituted tetrapeptide. It can be achieved. Merrifield's solid phase technology was used to produce the subject polypeptides; see, for example, M. Bodanszky and M.A. Ondetti, Peptide Synthesis;
It is clearly contemplated that the classical technique described in Interscience, 1966 may also be used. The identity and purity of the subject peptides were determined by well known methods such as thin layer chromatography, electrophoresis, amino acid analysis, etc. The invention is illustrated by the following examples, but is not limited thereto. In these examples and throughout the specification, parts are by weight unless otherwise specified. EXAMPLES In the preparation of the polypeptides of the invention, the following materials were obtained commercially: Alpha-BOC-L-glutamine-o-nitrophenyl-ester Alpha-BOC-ε-2-chloro-benzyloxycarbonyl-L- Lysine Alpha-BOC-O-benzyl-L-serine Alpha-BOC-L-alanine. In these reagents, BOC is t-butyloxycarbonyl. "Sequenal" class reagents, dicyclohexyl carbodiimide, fluorescamine, and resins for quantification of amino acid sequences were also commercially available. The resin used was a 200-400 mesh polystyrene divinylbenzene resin containing 1% divinylbenzene and 0.75 mmol chloride/g resin. In the production of polypeptides, 2 mmol of α
-BOC-L-glutamine was esterified to 2 mmol of chloromethylated resin in absolute alcohol containing 1 mmol of triethylamine at 80° C. for 24 hours. The resulting amino acid resin ester was filtered, washed with absolute alcohol, and dried. after that,
The other amino acid was coupled in the same correct order to the deprotected α-amino group of the peptide resin using an equivalent amount of dicyclohexyl carbodiimide to produce the polypeptide of the invention. After each conjugation, an aliquot of the resin was tested with a fluorescent amine and if positive fluorescence was found, the conjugation was considered incomplete and repeated with the same protected amino acid. As a result of several coupling reactions, an intermediate tetrapeptide-resin was produced. The peptide-resin was cleaved in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride for 60 minutes at 0°C with 1.2 ml of anisole as a scavenger per gram of the peptide-resin and the protecting groups was removed. The peptide mixture is washed with anhydrous ester and extracted with aqueous acid. The extract was lyophilized and the peptides were chromatographed on P-6Bio-Gel in 1N acetic acid. The resulting polypeptide was quantified to be 94% pure and determined to have the following order: H-ALA-LYS-SER-GLN-OH. I went like this. Thin layer chromatography was performed on 30 μg of sample on silica gel (Brinkman with fluorescent indicator).
Silica Gel, 20 x 20 cm, 0.1 mm thickness) using the following eluent: R _f ¹ : n-butanol: pyridine: acetic acid: water;
30:15:3:12 R _f ² : Ethyl acetate: Pyridine: Acetic acid: Water; 5:
5:1:3 R _f ³ : ethyl acetate: n-butanol: acetic acid: water;
1:1:1:1. Electrophoresis was performed on 100 μg of sample on Whatman 3 mm paper (11.5 x 56.5 cm).
A 5.6 pyridine-acetate buffer solution and a potential of 1000 volts were used for 1 hour. The spray reagents for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin. The following results were obtained: R _f ¹ = immobile, R _f ² = immobile,
and R _f ³ =0.336. Electrophoresis is 9.4cm towards the cathode.
caused the movement of Test Example To determine the activity and properties of the tetrapeptide produced in the example, the following chicken challenge assay was used. This test was performed by Brand, et al. Science, 193,
319-321 (July 23, 1976) and the references contained therein. Newly hatched chicken bone marrow was chosen as the inducible cell source. This is because it lacks appreciable numbers of Bu-1 ⁺ or Th-1 ⁺ cells. Cells pooled from the femurs and tibiotarsal bones of five newly hatched chickens of the line SC (Hy-Line) were deposited on a five-layer discontinuous bovine serum albumin (BSA) gradient by ultracentrifugation. It was separated into Cells from the two light layers were combined, washed, and suspended with the appropriate concentration of test polypeptide in RPMI 1630 medium at a concentration of 5 x 10 ⁶ cells/ml for culture. This medium contains 15 mmol hepes, 5% γ-
Globulin-free fetal bovine serum was supplemented with deoxyribonuclease (14-18 units/ml), heparin (5 units/ml), penicillin (100 units/ml) and streptomycin (100 μg/ml). Controls were cultured with BSA (1 μg/ml) or with medium alone. After culturing, cells were cultured in chicken C1 and guinea pigs as described in the reference paper.
C2-C9 complement fractions were used to test in the cytotoxicity assay. The proportion of Bu-1 ⁺ or Th-1 ⁺ cells in each layer is the cytotoxicity index, 100(a-b)/a, where a
and b are the percentage of live cells in the complement control and test formulations, respectively). The percentage of cells induced is determined by the induction agent (usually 1
~3%) was obtained by subtracting the mean value in the control culture from the mean value in the test culture. The specificity of the action of the test polypeptide and its similarity to ubiquitin demonstrated that the test polypeptide induced an increase in Bu-1 ⁺ B cells and Th- cells when ubiquitin was added at a concentration of 100 μg/ml.
Evidenced by induction of ¹⁺ T cells. This high dose of ubiquitin inactivates ubiquitin receptors and thus prevents cell induction by drugs acting through these receptors. As a result of this assay, the tetrapeptide of the example was
It was discovered that it exhibited biological activity similar to that of ubiquitin at ng/ml concentrations in inducing differentiation of both Th-1 ⁺ T and Bu-1 ⁺ B lymphocytes. Test Example A The test example assay was repeated using one of the following as the test peptide: 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 was observed in . B The test example assay was repeated using one of the following as the test peptide: H-SAR-LYS-D-ALA-GLN- _NH2H -SAR-LYS-SAR- _GLN -NH2HD- ALA-LYS-D-ALA-GLN-
In _each case, a biological activity similar to that of ubiquitin was observed. For the first of these polypeptides, this activity was observed at concentrations ranging from about 1 pg/ml to about 100 pg/ml. For the second polypeptide, activity was observed at concentrations as low as 0.1 pg/ml. EXAMPLE - Using the reaction techniques described above for the preparation of substituted polypeptides, polypeptides with the following formula are prepared: R-X-LYS-Y-GLN-R' These peptide amides are prepared on benzhydrylamine resin It was produced by solid phase synthesis methods known in the art. TABLE The polypeptides produced in Examples retain biological activity as described herein for active polypeptide segments. For confirmation, thin layer chromatography and electrophoresis were performed as follows. Thin layer chromatography was performed on 20 μg of sample using silica gel (Kieselgel, 5 x 20 cm) and n-butanol:acetic acid:ethyl acetate:water (1:1:1:1 ratio) (R _f ¹ ) as the eluent. , cellulose 6064 (Eastman, 20x20 cm) and n-butanol:pyridine:acetic acid:water (ratio ¹⁵ :10:3:12) ( _Rf2 ) as eluent. Electrophoresis was performed on a 50 μg sample using a Whatman
It was carried out on No. 3 paper (5.7 x 55 cm) using a pyridine-acetate buffer solution of PH 5.6 and a potential of 1000 volts for 1 hour. Spray liquids for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin. The following results were obtained (both values of R _f and electrophoresis were H-ARG-LYS-ASP-VAL-TYR
-OH): Table Following the thin layer chromatography and electrophoresis method of Example 1, the following results are obtained for the example compound: R _f ¹ = (0.155), R _f ² = immobile; R _f ³ =
0.265 and electrophoretic migration towards the cathode
It is 13.1cm. EXAMPLE - Using the reaction techniques described above for extending a polypeptide chain containing an active amino acid sequence, but substituted with terminal amino and carboxyl groups R and R', a polypeptide of the formula R-ALA-LYS-SER- A polypeptide of GLN-R' is provided, and a polypeptide substituted with the amino acids listed in the table below is produced. Example R R' GLN OH SAR OH The polypeptide derivatives produced in Example - retain biological activity as described herein for active polypeptide segments. Thin layer chromatography and electrophoresis of the example and the compounds in the example give the following results: TABLE TEST EXAMPLE To further illustrate the utility of the subject polypeptide, this example A microculture assay is described to estimate the number of cytotoxic lymphocytes generated upon stimulation with allograft antigens. The frequency of cytotoxic precursors between control animals and animals injected with various concentrations of drug was compared by limiting dilution assay. Materials and Methods Mouse Inbreed C57BL/6J (female,
8 weeks) at the Jackson Institute (Jackson Institute)
Laboratory, Bar Harbor, Maine). Inbreed DBA/2J (male or female) was also obtained from the Jackson Laboratory. Medium Phosphate buffered saline (PBS), RPMI1640,
Fetal bovine serum (FCS) - (lot number R776116),
N-2-hydroxyethylpiperazine-N'-2
- Ethanesulfonic acid (HEPES) buffer was obtained from Gibo (Grand Island); 2-mercaptoethanol was obtained from Eastman Kodak (Rochester, NY). Cells were washed with PBS and 10% FCS, 10 mmol HEPES buffer and 5 x 10 ^-5 mol 2
- Cultured in RPMI containing mercaptoethanol. Drug Treatment Test animals (C57BL/6J) were treated with various concentrations of H-SAR-LYS-SAR-GLN- in a volume of 0.2 ml.
After 1 and 24 hours of injection (intravenously or intraperitoneally) with NH ₂ (drug; label number G040) they were sacrificed for examination. Cell Preparation Cell suspensions from the spleens of C57BL/6J or DBA/2J mice were prepared by cutting the organs into small pieces and passing them through a wire mesh (60 gauge) into a 5 c.c. syringe plunger in a falcon Petri dish (falcon3002, 15
×60 mm). The cell suspension was allowed to stand at room temperature to allow large pieces of tissue to settle. The cell suspension was then transferred to a 15 ml Corning centrifuge tube and spun for 10 minutes at 1500 RPM in a Beckmen TJ-6 centrifuge. PBS all cell suspensions
I washed it at least three more times. After the third wash, responder (C57BL/6J) cells were resuspended in the medium and counted in an automatic hemocytometer (Coulter counter). DBA/2J (stimulator cells) during RPMI
Resuspend to 10 ⁷ cells/ml. 30 μg of mitomycin C was added to each ml of DBA/2J splenocytes and the mixture was incubated at 37° C. for 30 minutes. Mitomycin C
After treatment, splenocytes were washed three times with PBS to remove excess mitomycin C. DBA cells were then resuspended in medium and counted on an automatic hemocytometer. Mixed Lymphocyte Culture (MLC) MLC was cultured in microtiter trays (Linbro Chemicals, New Haven, Conn.).
IS-MVC-96). Each tray is 96-V
Contains a bottom reservoir. The outer reservoir surrounding the edge of the plate was not used for cell culture, but was filled with PBS.
to avoid evaporation from the culture reservoir. Sixty samples were prepared in each V-bottom tray. Typically each tray contains three concentrations of responder cells (20 replicates each)
and one concentration of stimulator cells. Responder cells were normally suspended at concentrations of 7.5×10 ⁵ , 5×10 ⁵ and 2.5×10 ⁵ /ml, and 0.1 ml was added to each well. The stimulator cell concentrations used were 10 ⁶ , 2.5×10 ⁶ , 5×
10 ⁶ /ml and 0.1 ml was added to each well. The same stimulator cell concentration was used throughout all plates. Control plates containing only responder cells without stimulator cells were also prepared to estimate background stimulation by medium. Cells were cultured for 6 days at 37° C. in a humidity-controlled incubator containing 5% CO ₂ . Target Red Blood Cells The target red blood cells used for the cytotoxicity assay were the DBA mastocytoma cell line P815. This cell line was maintained by regular passage of DBA/2J mice. 5 x ¹⁰⁸ P815 cells were used each passage and tumor cells from the peritoneal cavity of the carrier were used 4-5 days after passage. Wash tumor cells from the peritoneal cavity three times with PBS,
It was then labeled with Cr ⁵¹ at a concentration of 10 μci/10 ⁷ cells. Labeling was performed for 1 hour at 37°C in a humidity-controlled incubator. Labeled cells were then washed three times with PBS to remove excess labeled material. Cytotoxicity Assay After 6 days of culture, 0.1 ml of medium was collected from each well.
The cell pellet was removed without disturbing it. Then, using an automated micropipette ( ^MLA pipette), 100 μ of target red blood cells containing ^2.5 Cloudy (new pipette tips must be used for each reservoir). The microtiter trays were then placed in Sorvall GLC-2B at room temperature.
Spin at 1000 RPM for 7 minutes. The trays were then incubated for 4 hours at 37°C. 100 microliters of the supernatant liquid was then removed into a gamma counter (Amershen 196271). These tubes were then counted in a Beckmen gamma counter (Beckmen 310). These tubes were typically counted for 1 minute. Determination of the frequency of cytotoxic lymphocyte (CLP) precursors Limiting dilution analysis is a test of total response or no response described by a Poisson probability distribution. The probability of non-response is the zero-order term P ₀ = e ^- 〓 ^N (where δ = frequency of CLP,
and N=number of lymphocytes per reservoir). Thus, a plot of the ratio of unresponsive medium versus the logarithm of the dose of cells will yield a straight line with a slope of -δ and a frequency of CLP. In the example, the background chromium release (spontaneous release) from 20 reservoirs containing immediately responder cells that did not contain stimulator cells was averaged. Test pools were scored as positive if their counts were greater than 2.07 standard deviations and greater than the mean spontaneous value (P<
0.05). Spontaneous cell lysis typically ranged from 9-15% of the total counts added into target red blood cells. According to Poisson's equation P ₀ = e ^- 〓 ^N , when P ₀ = e ^-1 = 0.37 (corresponding to 37% non-responsive cultures), δ = 1/N, thus corresponding to 37% non-responsive cultures. The reciprocal of the number of responding cells is the frequency of CLP. The number of cells per well and their corresponding values for percentage non-response were entered into the computer. The computer calculates an optimal regression line through these points and the number of cells per well corresponding to 37% unresponsive cultures. The reciprocal of that value is the frequency of CLP. Results The frequency of cytotoxic lymphocyte precursors for each stimulator cell concentration was plotted as a function of drug dose. The mean and standard deviation of 20 replicates were also calculated for comparison. The three stimulator cell concentrations used were 10 ⁵ (suboptimal stimulation) and 2.5×10 ⁵ (optimal stimulation).
and 5×10 ⁵ (superoptimal stimulation). The drug tested was approximately 1p in the presence of suboptimal stimulant concentrations.
g/mouse ~ approx. 100ng/mouse (approx. 50pg/Kg ~
It was found to promote the production of cytotoxic lymphocyte precursors at a concentration of approximately 5 μg/Kg body weight). The test drug therefore acts as an immunomodulator at these concentrations, increasing the immune response of the cells of the treated mice. The invention has been described in terms of certain preferred embodiments. However, the invention should not be considered to be limited to the embodiments described, as modifications will be obvious to those skilled in the art.

Claims (1)

[Claims] 1 The following sequence: R-X-LYS-Y-GLN-R' where X is ALA, D-ALA or SAR; Y is SER, D-ALA, SAR or 2-
Me-ALA; R is H, GLN or SAR
and R′ is OH or NH ₂ . 2 R is H, X is ALA, Y is SER, and R' is
The peptide according to claim 1, which is OH. 3 R is H, X is SAR, Y is D-ALA, and
The peptide according to claim 1, wherein R' is _NH2 . 4 R is H, X is SAR, Y is SAR, and R′ is
The peptide according to claim 1, which is _NH2 . 5. The peptide according to claim 1, wherein R is H, X is D-ALA, Y is D-ALA, and R' is _NH2 . 6 R is H, X is SAR, Y is 2-Me-ALA,
The peptide according to claim 1, wherein R' is _NH2 . 7 R is GLN, X is ALA, Y is SER, and
The peptide according to claim 1, wherein R' is OH. 8 R is SAR, X is ALA, Y is SER, and
The peptide according to claim 1, wherein R' is OH.