NZ189101A

NZ189101A - Polypeptides having the ability to induce differentiation of both th-1+ t-lymphocytes and bu-1+ b-lymphocytes;pharmaceutical compositions

Info

Publication number: NZ189101A
Application number: NZ189101A
Authority: NZ
Inventors: G Goldstein
Original assignee: Ortho Pharma Corp
Priority date: 1977-12-08
Filing date: 1978-12-06
Publication date: 1984-07-06
Also published as: NO149631C; DE2853002A1; NL7812004A; SE444687B; FI67368B; JPS5498719A; ES475857A1; FR2411174B1; FI67368C; IL56150A; GB2014581A; DK149595C; FR2411174A1; IE782423L; NO149631B; JPS6327360B2; CH642058A5; SE7812614L; YU288078A; GR65013B

Description

New Zealand Paient Spedficaiion for Paient Number 1 89101 life _M&ii|"i^j„iMjl^ Patents Form No. 5 PATENTS ACT 195 3 COMPLETE SPECIFICATION Improvements in or relating to a polypeptide We, ORTHO PHARMACEUTICAL CORPORATION, a corporation organised and existing under the laws of the State of New Jersey, United States of America, of Raritan, New Jersey, United States of America do hereby declare the invention for which //we pray that a Patent may be granted to »«/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 (followed by page la) Field of the Invention This invention relates generally to new polypeptide segments and polypeptides, to methods for the preparation thereof, and uses thereof.

Description of the Prior Art It is well-known that many polypeptides have been isolated from various tissues and organs (including the blood) of animals. Many of these polypeptides are related to immune function in the body, as, for example, the various immune globulins, the thymic hormone thymopoietin, and the like. Indeed, we have - isolated and syn thesized several of these polypeptides, as described -in United States Patents Nos. 4,002,602 and 4,002,740 as well as in several scientific articles.

Until about the past decade, little was known about the thymus, although it is now understood that the thymus is one of the organs principally responsible for immune function in mammals and birds. Despite keen interest in possible -functions of the thymus and early speculation and experimentation, little was known of the function of the thymus until recently. It is now realized, however, that the thymus is a compound organ with both epithelial (endocrine) and lymphoid (immunological) components and thus the thymus is involved in the immunity functions of the body. The thymus consists of an epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells originating in haemopoietic tissues, Goldstein, et al., The Human Thymus, Heinemann, London, 1969. Lymphocytes are differentiated within the thymus and leave as mature thymus-derived cells, called T cells, which circulate to the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation within the thymus appears to be mediated by secretions of the epithelial cells of the thymus.

It has been known for some time that the thymus is.connected with the immunity characteristics of the body and, therefore, great interest has been indicated in substances which have been isolated from the thymus. In 189101 this regard, there have been published in recent years a relatively large body of articles based on scientific work relating to materials which are present in bovine thymus. In fact, we have published a number of articles which relate to research in this area. Pertinent publications may be found, for example, in The Lancet, July 20, 1968, pp. 119-122; Triangle, Vol. II, 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. 72, pp. 11-15, 1975; Biochemistry, Vol. 14, pp. 2214-2218, 1974; Nature, Vol. 255, pp. 423-424, 1975.

A second class of lymphocytes having immune function are the B lymphocytes or B cells. These are differentiated in the Bursa of Fabricius in birds and by an as-yet-unidentified organ in mammals. T-cells and B-cells cooperate in many aspects of immunity. See, for example, articles by us in Science, 193, 319 (July 23, 1976) and Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLI, 5 (1977).

A nonapeptide material has recently been isolated from porcine serum by J. F. Bach, et al. and identified as "facteur thymique serique" (FTS). The isolation of this material and its structure are disclosed in C. R. Acad. Sc. Paris, t. 283 (November 29, 1976), Series D-1605 and Nature 266, 55 (March 3, 1977). The structure of this nonapeptide has been identified as GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN, where "GLX" represents either glutamine or pyroglutamic acid. The material where GLX is glutamine or pyroglutamic acid has been synthesized. In these articles, Bach disclosed that his nonapeptide FTS selectively differentiated T cells (and not B cells) by use of an E rosette assay. Bach, therefore, concluded that his material was a thymic hormone. 189101 Recently, a more thorough investigation of the activity of this differentiated both T cells and B cells and was, therefore, more like"ubiquitin-in its activity than thymopoietin. Brand, Gilmour and Goldstein, Nature, 269;597 (1977).

It has now been discovered that a synthesized 4-amino acid polypeptide segment of this FTS nonapeptide possesses many of the characteristics of the nonapeptide discussed in the above publications.

Accordingly the invention comprises a polypeptide having the capability of inducing the differentiation of both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes, said polypeptide having the following sequence: R-X-LYS-Y-GLN-R' wherein X and Y are each natural or non-natural amino acid residues selected from the group consisting of L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alanyl, L-seryl, sarcosyl, 2-methylalanyl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, D-alanyl, and D-seryl and R and R' are each selected from the groups consisting of: nonapeptide by us disclosed that FTS r r' Hydrogen OH C^-C^ alkyl C6~C12 arY1 NH NHR? N(R?) 2 2 C6"C20 alkary! C6"C20 aralk^1 alkanoyl C2~C7 alkenyl C2~C7 alkynyl GLN or GLY GLY-GLY-SER GLY-GLY 7 SAR GLY-GLY-SER-ASN 189101 wherein is alkyl, C2~C7 alkenyl, C2~C7 alkynyl, C6~C20 aryl, C6~C20 aralkYl' or C6~C20 alkaryl» provided that when R is GLN, R' is other than GLY-GLY-SER-ASN, and the pharmaceutically acceptable salts thereof; further provided that said polypeptide induce the differentiation of both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes in the chicken induction assay at a concentration of one ng/ml or less.

Also provided is a procedure for preparation of the polypeptide segments and polypeptides of the invention by solid phase peptide synthesis, as well as therapeutic compositions containing the polypeptides, and methods for administration thereof to humans and animals for effecting biological actions thereon.

Description of Preferred Embodiments As._„indicated above, this invention is concerned with new polypeptide segments and polypeptides having therapeutic value in various areas, therapeutic compositions and method for their use utilizing the polypeptides of this invention, and methods for manufacture thereof.

In the preferred embodiment of the present invention, there is provided a biologically active polypeptide segment which has the following amino acid sequence: I. -ALA-LYS-SER-GLN-.

Since the biological activity of the subject polypeptide segment is generally retained upon substitution of a natural or non-natural amino acid residue for either or both of: 1) alanyl in the first position; and 2) seryl in the third position, the preferred embodiment of the present invention further includes biologically active polypeptide segments which ha^^^^g^S^llowing amino acid sequence: IA. -X-LYS-Y-GLN- "out ii 3 3 1*891 wherein, X and Y are each selected from the group consisting of natural and non-natural alpha—amino carboxylic acid (hereafter "amino acid") residues. While it is believed that the large majority of such substitutions of 5 X and Y groups will allow retention of biological activity, it is possible that certain natural or non-natural amino acid residues will interfere with the folding of the molecule (as discussed more fully below) and thus substantially eliminate the biological activity. Such 10 activity-destroying substituents are specifically excluded from the scope of the present invention.

The substituents X and Y are preferably selected from such natural amino acid residues as L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alanyl, 15 L-seryl, and the like; and frcm such non-natural amino acid residues as sarcosyl, 2-methylalanyl, the D-forms of the L-amino acids listed above, and the like. Whether a particular substitution allows retention of the biological activity of the polypeptide may be readily established by testing 20 it for differentiation of Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes in the chicken induction assay described below. Compounds which are specifically active in nanogram (ng)/milliliter (ml) concentrations (one ng/ml or less) in this assay are considered to be biologically 25 active.

A list of natural amino acids may be found in many reference books, e.g., R. T. Morrison and R. N. Boyd, "Organic Chemistry", Allyn and Bacon, 1959, Chapter 33. In addition to the natural amino acids (which are those 30 found in proteins), there are also a number of so-called "non-natural" amino acids which are not found in proteins although they sometimes occur naturally as metabolic intermediates or the like. These non-natural amino acids may be the D-isomers corresponding to the optically active 35 (L-form) natural amino acids or they may be entirely different chemical entities such as sarcosine (N-methyl glycine) or 2-methylalanine mentioned above. Lists of such non-natural amino acids are also found in many reference works.

ORTIi 3 3-3- 189101 j oo ■> —>.

Ul The polypeptide segment indicated in the principal embodiment above as Formula IA must additionally contain terminal substituents on the 4-amino acid sequence, thus yielding the subject polypeptides. These terminal substituents must not substantially affect the biological activity of the active 4-amino acid segment, as measured by the ability to induce the differentiation of Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes in the chicken induction assay described below. The subject polypeptides may be described by the following general formula: II. R-X-LYS-Y-GLN-R1 wherein X and Y are as previously described and R and R' are substituents on the terminal amino group and terminal carboxyl group, respectively, of the peptide segment which, as described above, do not substantially affect the biological activity of the active peptide segment. Since the active tetrapeptide segment is contained within a longer sequence in the naturally-occurring material iso- tr." lated by Bach, it should be understood that the terminal amino and carboxylic acid groups are not essential to the biological activity of the tetrapeptide segment, as is the case for some polypeptides. It is, therefore, considered that the scope of the present invention includes not only those tetrapeptide segments which are substituted by H and OH respectively, but also those which are terminally substituted by one or more other functional groups which do not substantially affect the biological activity disclosed herein. It should be clearly understood, however, that the nonapeptide described by Bach, et al., is specifically excluded from the scope of the present invention.

From this statement, it will be understood that these functional groups include such normal substitution as acylation on the free amino group and amidation on the free carboxylic acid group, as well as the substitution of additional amino acids and polypeptides. In these aspects, the polypeptide segments of this invention appear to be highly unusual since they exhibit the same ■ORTII 333 }■ ■ ^ ^ : . ; 7 " w biological activity as the natural nonapeptide of which the active tetrapeptide segment forms a portion. It is believed, therefore, that the activity requirements of the molecule are generated by its stereochemistry, that 5 is, by the particular "folding" of the molecule. In this regard, it should be understood that polypeptide bonds are not rigid but flexible, and polypeptides may exist as sheets, helices, and the like. As a result, the entire molecule is flexible and will "fold" in a certain 10 way. In the present invention, it'has been discovered that the novel tetrapeptide segments probably "fold" in a similar manner to the corresponding tetrapeptide segment in the natural nonapeptide in that they exhibit the same biological characteristics. For this reason, 15 the tetrapeptide segments may be terminally substituted by various functional groups so long as the substituents do not substantially affect the biological activity or interfere with the natural "folding" of the molecule.

The ability of the molecule to retain its 20 biological activity and natural folding is clearly illustrated by the fact that the tetrapeptide segments of this invention exhibit the same biological characteristics as the natural 9-amino acid peptide disclosed as FTS by J. F. Bach in the above disclosed articles. In this 25 nonapeptide, one tetrapeptide sequence of this invention may be identified within the molecule but only in combination with the other amino acids described therein.

Since the tetrapeptide segments of this invention provide the same biological activity as the nonapeptide FTS, 30 it is clear that the amino acids and peptide chains substituted on the terminal amino acid residues of the tetrapeptide segment do not affect the biological characteristics thereof.

In view of this discussion, it will, therefore, 35 be understood that R and R' in Formula II can be any substituent that does not substantially affect the biological activity of the active segment. Thus, for purposes of illustration R and R' may be any of the following substituents: . 1. - 189101 R R' Hydrogen OH C1-C7 alkyl NH2 Cg-C^2 aryl NHR^ ^6_("2o a^kary^ N(R7)2 aralky1 0R7 C^-C^ alkanoyl C2~C7 alkenyl C2~C-j alkynyl wherein R^ is C^-C^ alkyl, C^-Cj alkenyl, G> -C^ alkynyl, C6~C20 ary1' C6~C20 alkarY1' or C6~C20 aralkY1 • As pointed out above,-however, R and R' can also be neutral amino acid groups or residues of polypeptide chains having 2 to 20 carbon atoms. The following are illustrative: R *L!_ GLN GLY SAR GLY-GLY GLY-GLY-SER GLY-GLY-SER-ASN provided that, when R is GLN, R' is other than GLY-GLY-SER-ASN.

One preferred embodiment of the invention is that wherein X is L-alanyl, Y is L-seryl, R is hydrogen and R' is OH. This preferred embodiment may be symbolized chemically as: H2N-CH-CONH-CH-CONH-CH-CONH-CH-COOH CH3 (ch2)4 ch2 (ch2)2 nh2 oh conh2 h- ala - lys — ser — gln-oh a second preferred embodiment is that wherein X and y are each selected from the group consisting of sarcosyl, D-alanyl, and 2-methylalanyl; a more preferred embodiment being that wherein X is sarcosyl, Y is sarcosyl or D-alanyl, r is hydrogen, and r' is nh2 Also included within the scope of the invention are the pharmaceutically acceptable salts of the polypeptides. As acids which are able to form salts with the polypeptides, there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thio-cyanic acid, sulfuric acid, phosphoric acid, etc. and organic ■OHTII 333 189101 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, cinnamic acid, naphthalenesulfonic acid 5 or sulfanilic acid, for instance.

Throughout, the present specification, the amino acid components of the peptide are identified by abbreviations for convenience. These abbreviations are as follows. The D-amino acids are indicated by placing "D" 10 before the abbreviation, e.g., D-alanine is represented by "D-ALA".

Abbreviated Amino Acid Designation 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-Me t hy 1 a 1 an i'ne 2-Me-ALA The polypeptides of this invention are 4-amino acid peptides (and their substituted derivatives) which have been found to exhibit characteristics similar to the f - 9-amino acid polypeptide FTS isolated from porcine blood 30 as disclosed in the above-referenced Bach, et al., articles. The peptides of this invention are particularly characterized in their ability to induce the differentiation of T-precursor cells as well as B-precursor cells. Certain of the subject polypeptides are active in a concentration 35 as low as one picogram (pg)/ml in the chicken induction assay discussed below.

It has been found that the polypeptides of this invention induce the differentiation of immunocyte-pre-cursor cells in vitro in the same way as the nonapeptides 40 disclosed by Bach. Thus, the polypeptides of this invention •» w have been found to induce the differentiation of both T-precursor cells, as measured by the acquisition of the thymic differentiation antigen Th-1 as well as B-pre-cursor cells, as measured by the acquisition of the 5 differentiation antigen Bu-1. Stated another way, the subject polypeptides have the capability of inducing differentiation of both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes.

It has also been found that the subject poly-10 peptides increase the capability of in vivo production of cytotoxic lymphocytes upon stimulation by allogenic antigens. That is, administration of the subject polypeptides to, e.g., rats, promotes the production of cytotoxic lymphocyte precursors as measured by an in vitro 15 assay of rat spleen cells. Since the generation of cytotoxic lymphocytes directly corresponds to the extent of graft rejection in allogenic graft vs. host reaction, the above finding is further support for the immunologic utility of the subject polypeptides. 20 To provide an understanding of the importance of the differentiating biological characteristics of the polypeptides of this invention, it should be noted that the function of the thymus in relation to immunity may be broadly stated as the production of thymus-derived cells, 25 or lymphocytes, which are called T cells. T cells form a large proportion of the pool of recirculating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homograft responses), as effector'cells. T cells, 30 however, do not secrete humoral antibodies. These antibodies are secreted by cells (termed B cells) derived directly from the. bone marrow independently of the thymic influence. However, for many antigens, B cells require the presence of appropriately reactive T cells before 35 they can produce antibodies. The mechanism of this process of cell cooperation is not yet completely understood.

From this explanation, it may be said that in operational terms, the thymus is necessary for the development of cellular immunity and many humoral antibody ■6RTIL- 333 ^ ■-* /H « W jj 11 responses and it affects these systems by inducing, within the thymus, the differentiation of haemopoietic stem cells to T cells. This inductive influence is mediated by secretions of the epithelial cells of the thymus, that 5 is, the thymic hormones.

Further, to understand the operation of the thymus and the cell system of lymphocytes, and the circulation of lymphocytes in the body, it should be pointed out that stem cells arise in the bone marrow and reach the 10 thymus by the blood stream. Within the thymus, stem cells become differentiated to immunologically competent T cells, which migrate to the blood stream and, together with B cells, circulate between the tissues, lymphatics, and the blood stream.

The cells of the body which secrete antibody (B cells) also develop from haemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated in an organ analogous to the thymus, called the Bursa of Fabricius. In mammals, no 20 equivalent organ has been discovered and it is thought that these cells differentiate within the bone marrow.

Hence, they are termed bone marrow-derived cells or B cells. The physiological substances dictating this differentiation remain completely unknown. 25 As pointed out above, the polypeptides of this invention are therapeutically useful in the treatment of humans and animals. Since the new polypeptides have the capability of inducing the differentiation of lymphopoietic stem cells originating in the haemopoietic tissues 30 to both thymus-derived lymphocytes (T cells) and immunocompetent B cells which are capable of involvement in the immune response of the body, the products of this invention are considered to have multiple therapeutic uses. Primarily, since the compounds have the capability of 35 carrying out certain of the indicated functions of the thymus, they have application in various thymic function and immunity areas. A primary field of application is in the treatment of DiGeorge Syndrome, a condition in which there is a congenital absence of thymus. Injection ORTII 33 3 1891 12 of one of the subject polypeptides, as further set out below, will overcome this deficiency. Another application is in agammaglobulinemia, which is due to a defect of the putative B cell differentiative hormone of the 5 body. Injection of one of the subject polypeptides will overcome this defect. Since the subject polypeptides are extremely active at low concentrations, they are useful in augmenting the collective immunity of the body in that they increase therapeutic stimulation of 10 cellular immunity and humoral immunity and are thereby useful in the treatment of diseases involving chronic infection in vivo, such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections, and the like. Further, the subject peptides are con-15 sidered to be useful in any area in which cellular or humoral immunity is an issue and particularly where there are deficiencies in immunity such as in the DiGeorge Syndrome mentioned above. Further, because of the charac- lr-- teristics of the polypeptides, they have in vitro use-20 fulness in inducing the development of surface antigens of T cells, in inducing the development of the functional capacity to achieve responsiveness to mitogens and antigens, and cell collaborativity in enhancing the ability of B cells to produce antibodies. They have ill vitro 25 .. usefulness in inducing the development of B cells as measured by the development of surface receptors for complement. The subject peptides are also useful in inhibiting the uncontrolled proliferation of lymphocytes which are responsive to ubiquitin (described in our 30 United States Patent No. 4,002,602). An important characteristic of the subject polypeptides is their in vivo ability to restore cells with the characteristics of T cells and also their in vivo ability to restore cells with the characteristics of B cells. They are, therefore, 35 useful in the treatment of relative or absolute B cell deficiencies as well as relative or absolute T cell de- co A ficiencies, whether or not these deficiencies are due to crs 3 deficiencies in the tissu'e differentiating into B cells or the ^ ««/ thymus, respectively, or to some other cause.

J* iN ^ £RTH 333- V oo OS -5 '■'n IN 13 189101 A further important property of the polypeptides of this invention is that they are highly active in very low concentrations. Thus, it has been found that the polypeptides are generally active in concentrations of 5 about 1 ng/ml, while certain strikingly potent polypeptides (H-SAR-LYS-D-ALA-GLN-NH2 and H-SAR—LYS-SAR-GLN-NHj) are active in concentrations ranging from 0.1 pg/ml. The carrier may be any of the well-known carriers for this purpose including normal saline solutions, preferably 10 with a protein diluent such as bovine serum albumin to prevent adsorptive losses to glassware at these low concentrations. The polypeptides of this invention are generally active at a range of above 1 yg/kg of body weight, while certain strikingly potent polypeptides are 15 active from 1 ng/kg of body weight. For the treatment of DiGeorge Syndrome, the polypeptides may be administered at a rate of 1 to - 100 yg/kg of body weight, while the strikingly potent polypeptides may be administered at a rate of, . 1 to 100 ng/kg of body 20 weight. Generally, the same range of dosage amounts may be used in treatment of the other conditions or diseases mentioned. While the above discussion has been given with respect to parenteral administration, it should be understood that oral administration is also possible at dosage 25 ranges generally 100 to 1000 times greater than those for injection.

The polypeptides of this invention were prepared using the concepts similar to those described by Merrifield as reported in Journal of American Chemical Society, 85, 30 pp. 2149-2154, 1963. The synthesis involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by covalent bonds to a solid resin particle. By this procedure, reagents and by-products were removed by filtration and the recrystallization of 35 intermediates were eliminated. The general concept of this method depends on attachment of the C-terminal amino acid of the chain to a solid polymer by a covalent bond and the addition of the succeeding amino acids one at a time in a stepwise manner until the desired sequence is assem-bled. Finally, the peptide is removed from the solid support and protective groups removed. This method provides »• QRTII 30-3 . y——. toy ' " /rr-v rf\ i> ' '! 'j '- \**y a 14 a growing peptide chain attached to a completely insoluble solid particle so that it is in a convenient form to be filtered and washed free of reagents and by-products.

The amino acids may be attached to any suitable polymer which merely has to be readily separable from the unreacted reagents. The polymer may be insoluble in the solvents used or may be soluble in certain solvents and insoluble in others. The polymer should have a stable 10 physical form permitting ready filtration. It must contain a functional group to which the first protected amino acid can be firmly linked by a covalent bond.

Various insoluble polymers suitable for this purpose are those such as cellulose, polyvinyl alcohol, polymeth-15 acrylate and sulfonated polystyrene but in the synthesis of this invention, there was generally used a chlorcmethylated copolymer of styrene and divinylbenzene. Polymers which are soluble in organic solvents while being insoluble in aqueous solvents may also be used. One such polymer 20 is a polyethylene/glycol having a molecular weight of about 20,000, which is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide synthesis is described in F. Bayer and M. Mutter, Nature 237, 512 (1972) and references contained therein. 25 The various functional groups on the amino acid which were active, but which were not to enter into the reactions, were protected by conventional protecting groups as used in the polypeptide art throughout the reaction. Thus, the functional group on lysine was pro-30 tected by protecting groups which could be removed on completion of the sequence without adversely affecting the polypeptide final product. In the synthesis fluorescamine was used to determine if coupling was complete by an indication of positive fluorescence (see 35 Felix, et al., Analyt, Biochem., 52, 377, 1973). If complete coupling was not indicated, the coupling was repeated with the same protected amino acid before depro-tection.

OR-TH.. £3->- f The C-terminal amino acid may be attached to the polymer in a variety of well-known ways. Summaries of methods for attachment to halomethyl resins are given in Horiki, et al., Chem Letters, pp 165-168 (1978) and 5 Gisin, Helv. Chim. Acta, 5j6, 1476 (1973) , and references given therein.

The general procedure involved initially esteri-fying L-glutamine, protected on its amino groups, to the resin in absolute alcohol containing an amine. The 10 coupled amino acid resin was then filtered, washed with alcohol and water and dried. The protecting group on the a-amino group of the glutamine amino acid (e.g., t-BOC, i.e., t-butyloxycarbonyl), was then removed. The resulting coupled amino acid resin, having the free amino group, was 15 then reacted with a protected L-serine, preferably alpha-t-BOC-O-benzyl-L-serine to couple the L-serine. The reactions were then repeated with protected L-lysine and L-alanine until the complete molecule was prepared. The sequence of reactions was carried out as follows: Resin a-R^-Gln-OH a-R^-Gln-Resin Remove a-amino protecting group H-Gln-Resin R, R, a-R-^-L-Ser-OH a-R^-Ser-Gln-Resin Remove a-amino protecting group H-Ser-Gln-Resin R.

R- a-R-^-Lys-OH ^3 "2^ a-R^-Lys-Ser-Gln-Resin Remove a-amino _ „ protecting group X\^ I 3 12^ H-Lys-Ser-Gln-Resin ■QRTII 333 189101 a-R, -Ala-OH R, R~ l3^ I2 ct-R^-Ala-Lys-Ser-Gln-Res in Remove all protecting f groups and resin H-Ala-Lys-Ser-Gln-OH In the above sequence of reactions R^ is a protecting group of the a-amino group and R2 and R^ are protecting groups on the reactive side chains of the L-serine and L-lysine, respectively, which are not affected or removed when R^ is removed to permit further reaction.

Preferably, in the above intermediate tetrapeptide resin, the term R^ stands for a protective grouping such as t-butyloxycarbonyl, R2 stands for benzyl or substituted benzyl (e.g., 4 chlorobenzyl), and R^ stands for substituted benzyloxycarbonyl (e.g., 2,6-di-chlorobenzyloxycarbonyl). The resin is any of the resins mentioned above as being useful in the process.

After the final intermediate was prepared, the peptide resin was cleaved to remove the R^, R2, and R^ protecting groups thereon and the resin. The protecting groups were removed by conventional means, e.g., by treatment with anhydrous hydrogen fluoride, and the resulting free peptide was then recovered.

As pointed out above, in conducting the process, it is necessary to protect or block the amino groups in order to control the reaction and obtain the products desired. Suitable amino protecting groups which may be usefully employed include salt formation for protecting strongly-basic amino groups, or urethane protecting substitutes such as £-methoxy benzyloxycarbonyl and t-butyl-oxycarbonyl. It is preferred to utilize t-butyloxycarbonyl (BOC) or t-amyloxycarbonyl (AOC) for protecting the a-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since the BOC and AOC (t-amyloxycarbonyl) protecting groups are readily removed following such reaction and prior to'the subsequent step (wherein such a-amino group itself undergoes reaction) QP.TH—* 3 a 17 189101 by relatively mild action of acids (e.g., trifluoro-acetic acid), which, treatment does not otherwise affect groups used to protect other reactive side chains. It will thus be understood that the a-amino groups may be protected by reaction with any material which will protect the amino groups for the subsequent reaction (s) but which may later be removed under conditions which will not otherwise affect the molecule. Illustrative of such materials are organic carboxylic acid derivatives which will acylate the amino group.

In general, any of the amino groups can be protected by reaction with a compound containing a grouping of the formula: 0 II R,— O — C — ^ is any grouping which will prevent the amino wherein R^, group from entering into subsequent coupling reactions and which can be removed without destruction of the molecule. Thus, R^ is a straight or branched chain alkyl or alkenyl group, • preferably of 1 to 10 carbon atoms, and preferably halo- or cyano-substituted; aryl, preferably of 6 to 15 carbons; cycloalkyl, preferably of 5 to 8 carbon atoms; aralkyl, preferably of 7 to 18 carbon atoms; alkaryl, preferably of 7 to 18 carbon atoms; or heterocyclic, e.g., 4-pyridyl. - The aryl, aralkyl and alkaryl moieties may also be further substituted as by one or more alkyl groups of 1 to about 4 carbon atoms. Preferred groupings for R^include t-butyl, t-amyl, tolvl, xylyl and benzyl. Highly preferred specific amino-pro-tecting groups include benzyloxycarbonyl; substituted benzyloxycarbonyl, wherein the phenyl ring is substituted by.one or more halogens, e.g., CI or Br, nitror loweralkoxy (having 1-6 carbon atoms therein) e.g. methoxy, or loweralkyl (having 1-6 carbon atoms therein); . t-butyloxycarbonyl; t-a_myloxy-carbonyl; cyclohexyloxycarbonyl; vinyligxycarbonyl; adamantyloxysarbopyl; bipl^nylisopropoxycarbohyl; and the like. 0£her protecting groups wkicli Q.an be used include 4-pyridyloxycarbonyl; phthaloyl, p-tolyl-sulfonyl, formyl and the like.

QRgil 333 18 1.8 In conducting the general process of the invention, the peptide is built by reaction of the free a-amino group with a compound possessing protected amino groups. For reaction or coupling, the compound being attacked is activated at its carboxyl group so that the carboxyl group can then react with the free a-amino group on the attached peptide chain. To achieve activation the carboxyl group can be converted to any reactive group such as an ester, anhydride, azide, acid chloride, or the like. Alternately, a suitable coupling reagent may be added during the reaction. Suitable coupling reagents are disclosed, e.g., in Bodanszky, et al. -.Peptide Synthesis, Interscience, second edition, 1976, chapter five, including^carbodiimides (e.g., dicyclchexyl-carbodiimide), carbonyldiimidazole, and the like.

It should also be understood that during these reactions, the amino acid moieties contain both amino groups and carboxyl groups and usually one grouping enters into 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 which will not substantially affect other protecting groups, e.g., the group on the epsiIon-amino of the lysine molecule. The preferred procedure for effecting this step is mild acidolysis, as by reaction at room temperature with trifluoroacetic acid.

As may be appreciated, the above-described series of process steps results in the production of the tetrapeptide of Formula III as follows: III. H-ALA-LYS-SER-GLN-OH This tetrapeptide contains one tetrapeptide segment of this invention necessary for biological activity. The substitution of a natural or non-natural amino acid residue for either or both of L-alanyl and L-seryl IZ JUU98I ■GHHI 333* /<r~> 19 5- _ may be effected by replacing either or both of alanine and serine by the appropriately protected natural or non-natural amino acid in the above synthetic scheme, thus yielding the tetrapeptide of the following formula: IIIA. H-X-LYS-Y-GLN-OH wherein X and Y are as previously described. The substituted tetrapeptide of Formula II, wherein the terminal amino acid groups may be further substituted as described above, may then be prepared by reaction of the tetra-10 peptide of Formula (IIIA) or the protected peptide resin precursor with suitable reagents to prepare the desired derivatives. Reactions of this type such as acylation, esterification, amidation and the like, are, of course, well-known in the art. Further, other amino acids, that 15 is amino acid groups which do not affect the biological activity of the tetrapeptide sequence, may be added to either end of the peptide chain by the same sequence of reactions by which the tetrapeptide itself was synthesized. Still further, substitution for either or both the ala-20 nine or the serine moieties may be accomplished by employing the desired substituent (suitably protected) in place of alanine or serine in the preceding sequence of reactions by which the unsubstituted tetrapeptide was synthesized.

While the solid phase technique of Merrifield has been used to prepare the subject polypeptides, it is clearly contemplated that classical techniques described in, for example, M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience, 1966, may also be employed. 30 Identity and purity of the .subject peptides were determined by such well-known methods as thin layer chromatography, electrophoresisf amino acid analysis, and the like.

I O " 1 " " The following Examples are presented to illustrate the invention, but it is not to be considered as limited thereto. In the Examples, and throughout the specification, parts are by weight unless otherwise 5 indicated.

EXAMPLE I In preparation of the polypeptide of this invention, the following materials were purchased commercially: Alpha-BOC-L-Glutamine-o-nitrophenyl-ester Alpha-BOC-e-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-O-benzyl-L-serine Alpha-BOC-L-Alanine.

In these reagents, BOC is t-butyloxycarbonyl. 15 "Sequenal" grade 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, 200-400 mesh size containing 1% divinyl benzene and .75 mM of chloride 20 per gram of resin.

In preparation of the polypeptide, 2 mmoles of a-BOC-L-Glutamine were esterified to 2 mmoles of chloro-methylated resin in absolute alcohol containing ImM triethylamine for 24 hours at 80°C. The resulting amino acid 25 resin ester was filtered, washed with absolute alcohol and dried. Thereafter, the other a-BOC-amino acids were similarly coupled to the deprotected a-amino group of the peptide-resin in the correct sequence to result in the polypeptide of -this invention using equivalent amounts 30 of dicyclohexyl carbodiimide. After each coupling reaction, an aliquot of resin was tested with fluorescamine and if positive fluorescence was found, coupling was taken to be incomplete and was repeated with the same protective amino acid. As a result of the several coupling 35 reactions, the intermediate tetrapeptide-resin was prepared.

This peptide-resin was cleaved and the protective groups removed in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at OJftgH 333 c r n -• ^ fc V..-- J 2i - --*J 0°C for 60 minutes with 1.2 ml anisole per gram peptide-resin as scavenger. The peptide mixture was washed with anhydrous ether and extracted with aqueous acid. The extract was lyophilized and the peptide was chromatographed 5 on P-6 Bio-Gel in 1 N acetic acid. The resulting polypeptide was determined to be 94% pure and was determined to have the following sequence: H-ALA-LYS-SER-GLN-OH For identification, thin layer chromatography 10 and electrophoresis were performed as follows.

Thin layer chromatography was performed on a 30 yg sample on silica gel (Brinkman Silica Gel with fluorescent indicator, 20 x 20 cm, 0.1 mm thick) using the following eluents.

Rf^": n-butanol: pyridine : acetic acid:water; 30:15:3:12 2 R^ : ethyl acetate:pyridine:actxc acid:water; 5:5:1:3 R 3 f : ethyl acetate:n-butanol:actio acid:water; 1:1:1:1 Electrophoresis was performed on 100 yg sample on Whatman 3 mm paper (11.5 x 56.5 cm) using a pH 5.6 20 pyridine-acetate buffer solution and 1000 volts potential for one hour.

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

The following results were obtained: R-^" = 2 3 immobile, R^ = immobile, and R^ = 0.336. Electrophoresis resulted in a migration of 9.4 cm toward cathode.

EXAMPLE II To determine the activity and characteristics of the tetrapeptide produced in Example I, the following 30 chicken induction assay was employed. This assay is described in greater detail in Brand, et al., Science, 193 319-321 (July 23, 1976) and references contained therein.

Bone marrow from newly-hatched chickens was selected as a source of inducible cells because it lacks 35 an appreciable number of Bu-1+ or Th-1+ cells. Pooled ORTH 333 fP ^ ^ ^ ^ J cells from femur and tibiotarsus of five newly-hatched chicks of strain SC CHy-Line) were fractionated by ultra-centrifugation on a five-layer discontinuous bovine serum albumin (BSA) gradient. Cells from the two lighter layers 5 were combined, washed, and suspended for incubation at a concentration of 5 x 10^ cells per milliliter with the appropriate concentration of test polypeptide in RPMI 1630 medium supplemented with 15 mM hepes, 5 percent y-globulin-free fetal calf serum, deoxyribonuclease (14 to 10 18 unit/ml), heparin (5 unit/ml), penicillin (100 unit/ml), and streptomycin (100 yg/ml). Controls were incubated with BSA (1 yg/ml) or medium alone. After incubation, the cells were tested in the cytotoxicity assay using chicken CI and guinea pig C2 to C9 complement fractions as described in the reference article. The proportion 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 percentages of viable cells in the complement control and test preparation, respectively. The percentage of cells 20 induced was obtained by subtracting the mean values in the control incubations without inducing agents (usually 1 to 3 percent) from those of the test inductions.

The specificity of the action of the test polypeptide and its similarity to ubiquitin were demonstrated 25 by the. inhibition of induction of Bu-1+ B cells and Th-1+ T cells by the test polypeptide upon addition of 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 which acts through 30 these receptors.

As a result of this assay, it was discovered that the tetrapeptide of Example I displayed biological activity similar to that of ubiquitin in inducing the differentiation of both Th-1+ T and Bu-1+ B lymphocytes in 35 ng/ml concentrations. 6 W ^ i J 23 " ^ EXAMPLE III A. The assay of Example II was repeated, using as the test polypeptide one of the following: H-GLN-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN-OH 5 H-GLN-ALA-LYS-SER-GLN-OH H-SAR-ALA-LYS-SER-GLN-OH In each case, biological activity similar to that of ubiquitin was observed.

B. The assay of Example II was repeated, using 10 as the test polypeptide one of the following: h-sar-lys-d-ala-gln-nh2 h-sar-ly s-sar-gln-nh 2 h-d-ala-lys-d-ala-gln-nh2 In each case, biological activity similar to that of 15 ubiquitin was observed. For the first of these polypeptides, this activity was observed in the range of concentration from about 1 pg/ml to about 100 pg/ml. For the second polypeptide, activity was observed at a concentration as low as 0.1 pg/ml.

EXAMPLES IV - VI Using the reaction techniques described hereinabove for preparing substituted polypeptides, these are prepared polypeptides of the following formula: r-x-lys-y-gln-r' These peptide amides were prepared on a benzhydrylamine resin by solid phase synthesis techniques known in the art.

EXAMPLE number r x Y r' iv h sar d-ala nh2 iva h sar sar nh2 v h d-ala d-ala nh2 va h d-ala sar nh2 vi h sar 2-me-ala nh2 via h sar sar oh 24 The polypeptides prepared in Examples IV-VI retain the biological activity as described herein for the active polypeptide segment.

For identification, thin layer chromatography 5 and electrophoresis were performed as follows.

Thin layer chromatography was performed on 20 yg samples on silica gel (Kieselgel, 5 x 20 cm) using as eluent n-butanol:acetic acid:ethyl acetate:water in proportions of 1:1:1:1 (R^) and on cellulose 6064 (Eastman, 10 20 x 20 cm) using as eluent n-butanol:pyridine:acetic acid: 2 water in proportions of 15:10:3:12 (Rf ).

Electrophoresis was performed on 50 yg samples on Whatman No. 3 paper (5.7 x 55 cm) using a pH 5.6 pyridine-acetate buffer solution and 1000 volts potential 15 for one hour. The compounds migrate toward the cathode.

The following results were obtained (both R^ values and electrophoresis are given relative to H-ARG-20 LYS-ASP-VAL-TYR-OH): , 2 Electrophoresis Example R^ R^ migration purity IV 0.44 0.68 2.07 98% IVA 0.88 0.60 1.78 98% V 0.56 0.71 2.10 98% Following the thin layer chromatography and electrophoresis procedure of Example I, the following results are obtained for the compound of Example VI: Rf^ = 2 3 (0.155), R^ = immobile, R^ = 0.265 and electrophoresis migration is 13.1 cm toward cathode.

EXAMPLE VII The tetrapeptide resins having protected LYS and SER prepared as in Examples I and VIA are each acylated by reaction with acetic anhydride under acetylating conditions, followed by removal of the protecting groups and the resin, 35 to prepare the following acylated derivatives: ch3co-ala-lys-ser-gln-oh ch3co-sar-lys-sar-gln-oh GRTH' 333 « - V ^ ^ EXAMPLE VIII The protected tetrapeptide resins prepared as in Examples I and VIA are each transesterified from the resin by reaction with sodium methoxide in methyl alcohol under 5 transesterification conditions, followed by removal of the protecting groups, to prepare the esterified derivatives of the following formulas: h-ala-lys-ser-gln-och3 H-SAR-LYS-SAR-GLN-OCH3 EXAMPLE IX The protected tetrapeptide resins prepared as in Examples I and VIA are each cleaved from the resin with diethyl amine under reaction conditions known in the art, followed by removal of the protecting groups, to prepare 15 the following amino substituted derivatives: h-ala-lys-ser-gln-n (cjhg) 2 h-sar-lys-sar-gln-n(c2h5)2 example x Following the methods of Examples I and VIA but 20 substituting for the ALA or SAR used to add the N-terminal amino acid residue, an equivalent amount of suitably protected N-a-ethyl-L-alanine or N—a-ethyl-sarcosine, respectively, there are prepared the following: c2h5-ala-lys-ser-gln-oh C2H5-SAR-LYS-SAR-GLN-OH example xi Cleaving the protected resin tetrapeptides formed in Example X from the resin using ammonia in dimethyl-formamide under amidation conditions, followed by removal 30 of the protecting groups yields peptide amides of the formulas: c2h5-ala-lys-ser-gln-nh2 C2H5-SAR-LYS-SAR-GLN-NH2 QEIS-^e- 26 ^ J •* example xii The protected acetylated tetrapeptide resins prepared as in Example VII are each reacted with, ammonia in dimethylformamide under amidation conditions, followed 5 by removal of the protective groups, to prepare the following peptide amides: ch3co-ala-lys-ser-gln-nh2 ch3co-sar-lys-sar-gln-nh2 examples xiii - xxvi 10 Using the reaction techniques described herein above for the lengthening of the polypeptide chain, the following polypeptides are prepared which contain the active amino acid sequence but which are substituted on the terminal amino and carboxylic groups R and R1 to 15 provide the polypeptide of formula: r-ala-lys-ser-gln-r' which is substituted by the amino acids given in the following Table as indicated. example number R r' xiii gln oh xiv sar oh xv E gly xvi h gly-gly xvii h gly-gly-ser xviii h gly-gly-ser-asn xix gln gly xx gln gly-gly xxi gln gly-gly-ser xxii sar gly xxiii sar gly-gly xxiv sar gly-gly-ser xxv sar gly-gly-ser-asn xxvi gln gly-gly-ser-asn The polypeptide derivatives prepared in Examples 35 iv-xxvi retain the biological activity as described herein for the active polypeptide segment. 189101 21 Following the thin layer chromatography and electrophoresis procedure of Example I for the compound of Examples XIII to XIV the following results are obtained: Example XIII XIV R. immobile immobile immobile immobile 0.303 0.186 electrophoresis migration toward cathode 7.4 cm 8.3 cm Following the thin layer chromatography and electrophoresis procedures of Examples IV-V for the compound of Example XXVI, the following results are ob- 12 . tamed: R^ = 0.23, R^ =0.25, electrophoresis migration toward cathode = 0.88.

EXAMPLE XXVII To further illustrate the utility of the subject polypeptides, this example describes a microculture assay tr." . for estimating the frequencies of the cytotoxic lymphocytes produced upon stimulation by allogenic antigens. The frequencies of cytotoxic precursors between control animals and animals injected with various concentrations of the drug were compared by a limiting dilution assay.

Materials and Methods Mice Inbreed C57 BL/6J (female, 8 weeks) were obtained from Jackson Laboratory, Bar Harbor, Maine.

Inbreed DBA/2J (male or female) 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-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer were obtained from Gibo, Grand Island; 2-mercaptoethanol was from Eastman Kodak, Rochester, N.Y. Cells were washed with PBS and cultured in RPMI containing 10% FCS, 10 mM HEPES buffer and 5x10 ^M 2-mercaptoethanol. e-RTH 33 Ih , • ' ;' -'N 2 8 u ^ ^ ■* Drug treatment The test animals (C57 BL/6J) were injected (i.v. or i.p.) with various concentrations of H-SAR-LYS-SAR-GLN-NH2 (the drug; identification no. G040) in 0.2 ml 5 volume 24 hours before they were sacrificed for the experiments.

Cell preparations Cell suspensions from spleens of C57 BL/6J mice or DBA/2J mice were prepared by mincing the organ and 10 pressing them through a wire mesh (60 gauge) with the plunger of a 5 c.c. syringe into a falcon petri dish (falcon 3002, 15x60 mm). The cell suspensions were allowed to stand at room temperature for 10 minutes to let the big chunks of tissue settle. The cell 15 suspensions were then transferred to 15 ml Corning centrifuge tubes (Corning 25310) and spun for 10 minutes at 1500 RPM in the Beckmen TJ-6 centrifuge.

All cell suspensions were washed at least three more times with PBS. After the third wash, the responder 20 (.C57 BL/6J) cells were resuspended in culture medium and counted in the Coulter counter. DBA/2J (stimu- 7 lator cells) were resuspended in RPMI to 10 cells/ ml. 30 yg of mitomycin C was added to each ml of the DBA/2J spleen cells and the mixtures were incubated 25 at 37° for 30 minutes. After 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 were set up in microtiter trays (Linbro Chemicals, New Haven, Conn., IS-MVC-96). Each tray contained 96-V bottom wells. The outside wells surrounding the edge of the plate were not used for cell culture but 35 filled with PBS to avoid evaporation from the culture wells. 60 samples were set up in each V-bottom tray. Usually each tray contained three responder cell concentrations (20 replicates of each) and one stimulator 9R$£L-3J3- ■ | ^ r ^ i ^ v ■ ' ; 29 « cell concentration. The responder cells were usually suspended to concentrations of 7.5x10 , 5x10 and 2.5x10^ per ml and 0.1 ml was added to each well. The 6 6 stimulator cell concentrations used were 10 , 2.5x10 , 5 5x10^ per ml, also 0.1 ml was added to each well. The same stimulator cell concentration was used throughout the whole plate. Control plate containing only responder cells with no stimulator cells was also set up for estimating background stimulation due to the 10 medium. The cells were cultured for six days at 37°C in a humidified incubator containing 5% CC^.

Target cells The target cell used in the cytotoxic assay was a DBA mastocytoma cell line P815. The cell line was main- o tained by routine passage through DBA/2J mice. 5x10 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 passage. The tumor cells from the peritoneal cavity were washed three 20 times with PBS and then labeled with Cr^1 at a con centration of 100 yci per 10^ cells. Labeling was done for an hour at 37°C in a humidified incubator. The labeled target cells were then washed for three times with PBS to remove any excess label.

Cytotoxic assay After six days of culture, 0.1 ml of medium was removed from each well without disturbing the cell pellet.

Then, using an automatic micropipet (MLA pipet), 100 4 51 yl of target cells, containing 2.5x10 Cr -labeled 30 target cells were pipetted into each well, resuspend- ing the cell pellet during the process (a new pipet tip has to be used for each well). The microtiter trays were then spun at room temperature at 1000 RPM for seven minutes in the Sorvall GLC-2B. The trays 35 were then incubated for four hours at 37°C. 100 micro liters of supernatant were then removed into Gamma counting tubes (Amershen 196271). The tubes were then counted in the Beckmen Gamma Counter (Beckmen 310). The tubes were usually counted for one minute.

QRTII 333 I' < . S 3 0 l: ^ - -j $ Determination of the frequencies of the precursors of cytotoxic lymphocytes (CLP) The limiting dilution analysis is an all or none response assay described by the Poisson probability distribution. The probability of a non-response is 5 given by the zero order term Po=e where <5 = fre quency of CLP and N = the number of lymphocytes per well. Thus a plot of the logarithm of the proportion of non-responding cultures vs cell dose should yield a straight line with a slope of -6, the frequency of 10 CLP.

In the Example, the background chromium release (spontaneous release) from 20 wells containing just responder cells (C57 BL/6J) with no stimulator cells were averaged. Test wells were scored as positive 15 if their counts were greater than the mean spon taneous value by more than 2.07 standard deviations (P<0.05). The spontaneous lysis usually ranged from 9-15% of the toal counts incorporated into the tar- —5N get cells. According to the Poisson equation Po=e , 20 when Po=e~"'' = 0.37 (corresponds to 37% non-responding cultures), 5 = 1/N, thus the reciprocal of the responding cell number corresponding to 37% non-responding cultures is the CLP frequency. Usually the number of cells per well and their corresponding value for per-25 cent non-responders were fitted into the computer which compute the best fit regression line through these points and the number of cells per well which correspond to 37% non-responding cultures, the reciprocal of that value is the frequency of the CLP.

Results The frequencies of cytotoxic lymphocyte precursors for each stimulator cell concentration were plotted as a function of drug dose. The mean and standard deviation of the twenty replicates were also computed for com- parison. The three stimulator cell concentrations 5 used were 10 (suboptimal stimulation), 2.5x10 (optimal stimulation) and 5x10^ (over optimal stimulation) . It was found that the test drug promoted 31 the production of cytotoxic lymphocyte precursors at concentrations of from about 1 pg/mouse to about 100 ng/mouse (equivalent to about 50 pg/kg to about 5 yg/kg body weight) in the presence of suboptimal stimulator cell concentrations. The test drug is therefore acting as an immuno-regulator at these concentrations to increase the cellular immune response of the treated mice.

The invention has been described herein with reference to certain preferred embodiments. However, as obvious variations will appear to those skilled in the art, the invention is not to be considered as limited thereto.

GRT-H—36-3" 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 1 2

Claims

WHAT WE CLAIM IS: WHAT ID CLAIMED IDl 32 189101

1. A polypeptide having the capability of inducing the differentiation of both Th-1*" T-lymphocytes and Bu-1+ B-lymphocytes, said polypeptide having the following sequence:

R-X-LYS-Y-GLN-R'

wherein X and Y are each natural or non-natural amino acid residues selected from the group consisting of L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alanyl, L-seryl, sarcosyl, 2-methylalanyl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, D-alanyl, and D-seryl and R and Rr are each selected from the groups consisting of:

R

Hydrogen C^-C^ alkyl

C6"C12 aryl

C6-C20

C6~C20 aralkyl cl"c7

alkenyl

C2-C7 alkynyl

GLN

SAR

Rr alkanoyl oh nh2

nhr?

ncr7)2

or-,

GLY

GLY-GLY

GLY-GLY-SER

GLY-GLY-SER-ASN

wherein R^ is C^~C7 alkyl, C2-C^ alkenyl, Gj-Cy alkvnyl,

C6~C20 arY-'-' C6~C20 araikyl' or C6~C20 alkarYl' provided that when R is GLN, R* is other than GLY-GLY-SER-ASN, and the pharmaceutically acceptable salts thereof; further provided that said polypeptide induce the differentiation of both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes in the chicken induction assay at a concentration of one ng/ml or less.

2. A polypeptide according to Claim 1, wherein R is hydrogen and R' is OH.

3. A polypeptide according to Claim 1, wherein R is CH^CO- and R1 is OH.

4. A polypeptide according to Claim 1, wherein R is CH^ and R' is OH.

5. A polypeptide according to Claim 1, wherein R is H and R* is NH0.

0-RTn'

189101

33

6. A polypeptide according to Claim 1, wherein

2 R is H and R' is N(C2H^)2.

7. A polypeptide according to Claim 1, wherein

2 R is CH^CO- and R' is NH2.

8. A polypeptide according to Claim 1, wherein

2 R is H and R' is -OCH^.

1 9 . A polypeptide according to Claim 1, wherein

2 R is C2H^ and R' is -NH2>

1 LD.' A polypeptide according to Claim 1, wherein

2 R is GLN and R' is -OH.

1 11. A polypeptide according to Claim 1, wherein

2 R is SAR and R' is -OH.

1 12. A polypeptide according to Claim 1, wherein

2 R is hydrogen and R1 is GLY.

1 13. a polypeptide according to Claim 1, wherein

2 R is hydrogen and R1 is GLY-GLY.

1 14- A polypeptide according to Claim 1, wherein

2 R is hydrogen and R' is GLY-GLY-SER.

15. A polypeptide according to Claim 1, wherein

2 R is hydrogen and R' is GLY-GLY-SER-ASN.

1 16. A polypeptide according to Claim 1, wherein

2 R is GLN and R' is GLY.

1 17. A polypeptide according to Claim 1, wherein

2 R is GLN and R' is GLY-GLY.

1 18. A polypeptide according to Claim 1, wherein

2 R is GLN and R' is GLY-GLY-SER.

- 33 -

2

3

1

2

3

4

5

7

8

9

10

11

12

13

14

15

16

GR'i'H -3 -3-3

18910

34

1

19.

A

polypeptide according to Claim

1, wherein

2

R

is

SAR

and

R'

is GLY.

1

20.

A

polypeptide according to Claim

1, wherein

2

R

is

SAR

and

R'

is GLY-GLY.

1

21.

A

polypeptide according to Claim

1, wherein

2

R

is

SAR

and

R1

is GLY-GLY-

SER.

1

22.

A

polypeptide according to Claim

1, wherein

2

R

is

SAR

and

R'

is GLY-GLY-

SER-ASN.

1

23.

A

polypeptide of the following sequence:

H-ALA-LYS-SER-GLN- OH

and the pharmaceutically acceptable salts thereof.

24. A polypeptide of the following sequence:

R-X-LYS-Y-GLN-R'

wherein X and Y are each selected from the group consisting of D-ALA and SAR and R and R' are each selected from the groups consisting of:

C6~C12 C6~C20

Hydrogen C^-C7 alkyl aryl alkaryl

C6~C20 aralky!

C^-C-y alkanoyl C2-C^ alkenyl C 2~C7 alkynyl gln SAR

r'

oh nh2

nhr?

n(r7)2

or?

gly gly-gly gly-gly-ser gly-gly-ser-asn wherein R7 is C1~C7 alkyl, C2~C7 alkenyl, -C7 alkynyl,

C6~C20 arylf C6~C20 aralkY1' 0;c C6~C20 alkarYl' Provided that when R is GLN, R' is other than GLY-GLY-SER-ASN, and the pharmaceutically acceptable salts thereof.

' 25. A polypeptide of the following sequence:

H-SAR-LYS-SAR-GLN-NH2 and the pharmtacg\itically acceptabl,^. s^lts thereof.

O A

1

2

3

1

2

3

4

1

2

3

4

1

2

3

4

1

2

3

4

CF-TH -33 3

35 169101

26 . a polypeptide of the following sequence:

h-sar-lys-d-ala-gln-nh2 and the pharmaceutically acceptable salts thereof.

27.. A therapeutic composition of matter comprising a therapeutically effective amount of a polypeptide of Claim 1 in a pharmaceutically acceptable carrier.

28 . A therapeutic composition" of matter according to Claim 27, wherein the therapeutically effective amount of the polypeptide ranges from 1 to

100 yg/kg body weight.

29 . A therapeutic composition of matter comprising a therapeutically effective amount of a polypeptide of Claim 24 in a pharmaceutically acceptable carrier.

30 • A therapeutic composition of matter comprising from 1.0 ... ng/kg to 10 0 ng /kg body weight of the polypeptide of Claim 25 or Claim 26 in a pharmaceutically acceptable carrier.

189101

31. A process for preparing a polypeptide of Claim 1 or a pharmaceutically acceptable salt thereof, substantially as hereinbefore described and with reference to the accompanying examples I and IV-XXV,.

32. A process for preparing a polypeptide according to Claim 1, wherein R is hydrogen and R' is OH, substantially as hereinbefore described and with reference to the accompanying examples I and VIA.

33. A process for preparing a polypeptide according to Claim 1, wherein R is CH^CO- and R1 is OH, substantially as hereinbefore described and with reference to the accompanying example VII.

34. A process for preparing a polypeptide according to Claim 1, wherein R is H and R' is , substantially as hereinbefore described and with reference to the accompanying examples IV, IVA, V, VA and VI.

35. A process for preparing a polypeptide according to Claim 1, wherein R is H and R' is N(C2H,-)2, substantially as hereinbefore described and with reference to the accompanying example IX.

i

3 1 JAM 1984 ,

I89101

36- A process for preparing a polypeptide according to

Claim 1, wherein R is CH^CO- and R' is NH2, substantially as hereinbefore described and with reference to the accompanying example XII.

37- A process for preparing a polypeptide according to Claim 1, wherein R is H and R' is -OCH^, substantially as hereinbefore described and with reference to the accompanying example VIII

38. A process for preparing a polypeptide according to Claim 1, wherein R is C2H5 and R' is -NH2, substantially as hereinbefore described and with reference to the accompanying example XI.

39.. A process for preparing a polypeptide according to Claim 1, wherein R is GLN and R' is -OH, substantially as hereinbefore described and with reference to the accompanying example XIII.

40. A process for preparing a polypeptide according to Claim 1, wherein R is SAR and R' is -OH, substantially as hereinbefore described and with reference to the accompanying example XIV.

41. A process for preparing a polypeptide according to Claim 1, wherein R is hydrogen and R' is GLY, substantially as hereinbefore described and with reference to the accompanying example XV.

42. A process for preparing a polypeptide according to Claim 1, wherein R is hydrogen and R' is GLY-GLY, substantially as hereinbefore described and with reference to the accompanying example XVI.

43. A process for preparing a polypeptide according to Claim 1, wherein R is hydrogen and R' is GLY-GLY-SER, substantially as hereinbefore described and with reference to the accompanying example XVII. . 1 V

189101

44. A process for preparing a polypeptide according to Claim 1, wherein R is hydrogen and R1 is gly-gly-SER-ASN, substantially as hereinbefore described and with reference to the accompanying example XVIII.

45. A process for preparing a polypeptide according to Claim 1, wherein R is GLN and R1 is GLY, substantially as hereinbefore described and with reference to the accompanying example XIX.

46. A process for preparing a polypeptide according to Claim 1, wherein R is GLN and R' is GLY-GLY, substantially as hereinbefore described and with reference to the accompanying example XX.

47 . A process for preparing a polypeptide according to Claim 1, wherein R is GLN and R' IS GLY-GLY-SER, substantially as hereinbefore described and with reference to the accompanying example XXI.

48 . A process for preparing a polypeptide according to Claim 1, wherein R is SAR and R' is GLY, substantially as hereinbefore described and with reference to the accompanying example XXII.

49 . A process for preparing a polypeptide according to Claim 1, wherein R is SAR and R' is GLY-GLY, substantially as hereinbefore described and with reference to the accompanying example XXIII.

50. A process for preparing a polypeptide according to Claim 1, wherein R is SAR and R1 is GLY-GLY-SER, substantially as hereinbefore described and with reference to the accompanying example XXIV.

189101

51 . A/ process for preparing a polypeptide according to

Claim 1, wherein R is SAR and R1 is GLY-GLY-SER-ASN, substantially as hereinbefore described and with reference to the accompanying example XXV.

52 _ A process for preparing a polypeptide of Claim 24 or a pharmaceutically acceptable salt thereof, substantially as hereinbefore described and with reference to the accompanying examples IV, IVA, V, VA, VIA and VII-XII.

53 . A process for preparing a polypeptide of the following sequence: H-SAR-LYS-SAR-GLN-NI^ or a pharmaceutically acceptable salt thereof, substantially as hereinbefore described and with reference to the accompanying example IVA.

54. A process for preparing a polypeptide of the following sequence: H-SAR-LYS-D-ALA-GLN-NH2 or a pharmaceutically acceptable salt thereof, substantially as hereinbefore described and with reference to the accompanying example IV.

5.5 . . A polypeptide of Claim 1 or a pharmaceutically acceptable salt thereof, whenever prepared according to the process claimed in Claim 31.

56 . A polypeptide according to Claim 1, wherein R is hydrogen and R1 is OH, whenever prepared according to the process claimed in Claim 32"-.

57 . A polypeptide according to Claim 1, wherein R is CH^CO- and R' is OH, whenever prepared according to the process claimed in Claim 33 •

58 . A polypeptide according to Claim 1, wherein R is H and R' is NH2, whenever prepared according to the process claimed in

Claim 34

3 1JAK;?34

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59 • A polypeptide according to Claim 1, wherein R is H and R' is N(C2H^)2» whenever prepared according to the process claimed in Claim 35 .

60. A polypeptide according to Claim 1, wherein R is CH^CO- and R' is NH2, whenever prepared according to the process claimed in Claim 36 •

61* A polypeptide according to Claim 1, wherein R is H and R' is -OCH^, whenever prepared according to the process claimed in Claim 3 62. A polypeptide according to Claim 1, wherein R is C^H,. and R' is "NH^, whenever prepared according to the process claimed in Claim 381.

6 3. A polypeptide according to Claim 1, wherein R is GLN and R' is -OH, whenever prepared according to the process claimed in Claim 39. 6 4. A polypeptide according to Claim 1, wherein R is SAR and R1 is -OH, whenever prepared according to the process claimed in Claim 40.

65. A polypeptide according to Claim 1, wherein R is hydrogen and R1 is GLY, whenever prepared according to the process claimed in Claim 41 .

66. A polypeptide according to Claim 1, wherein R is hydrogen and R' is GLY-GLY, whenever prepared according to the process claimed in Claim 42-

67. A polypeptide according to Claim 1, wherein R is hydrogen and

R' is GLY-GLY-SER, whenever prepared according to the process claime< in Claim 43.*

68- A polypeptide according to Claim 1, wherein R is hydrogen and R1 is GLY-GLY-SER-ASN, whenever prepared according to the process claimed in Claim 44. 0

I 29101

69., A polypeptide according to Claim 1, wherein R is GLN and R' is GLY, whenever prepared according to the process claimed in Claim 45.

70.- A polypeptide according to Claim 1, wherein R is GLN and R' is GLY-GLY, whenever prepared according to the process claimed in Claim 46.

71. A polypeptide according to Claim 1, wherein R is GLN and R' is GLY-GLY-SER, whenever prepared according to the process claimed in Claim 47.

72. A polypeptide according to Claim 1, wherein R is SAR and R' is GLY, whenever prepared according to the process claimed in Claim 48.

73* A polypeptide according to Claim 1, wherein R is SAR and R'

1

is GLY-GLY, whenever prepared according to the process claimed in Claim 49.

74. A polypeptide according to Claim 1, wherein R is SAR and R* is GLY-GLY-SER, whenever prepared according to the process claimed in Claim 5P.

75. A polypeptide according to Claim 1, wherein R is SAR and R' is GLY-GLY-SER-ASN, whenever prepared according to the process claimed in Claim 51-

7 6.. A polypeptide of Claim 2 4 or a pharmaceutically acceptable salt, thereof, whenever prepared according to the process claimed in Claim 52.

77. A polypeptide of the following sequence: H-SAR-LYS-SAR-GLN-NI^ or a pharmaceutically acceptable salt thereof, whenever prepared according to the process claimed in Claim 53.

-r*

-41- ? .

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IS- A polypeptide of the following sequence: H-SAR-LYS-D-ALA-GLN-NH2 or a pharmaceutically acceptable salt thereof, whenever prepared according to the process claimed in Claim 54.

WEST-WALKER, McCABE

per:

ATTORNEYS FOR THE AF;?L!CA.MT

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