AU602483B2 - Immunoregulatory peptides - Google Patents

Description

AU-Al 5 3 1 9 8 /8 6 pm R. '?P13 VOR 4UZ WORLD INTELLE+cL ~~yld PC iBuru.0 INTERNATIONAL APPLICATION PUBLISH-YhUNDER THE PATENT COOPERATION TREATY (PCT) 1) Internatio~oal Patent Classification 4 (11) Internatinal Publication Number: WO 86/ 04334 C07K 5/00, 7/00, A61K 37/02 Al (43) Intern?,ional Publication Date: 31 July 1986 (3 1.07.86) (21) International Application Number: PCT/EP86/00012 (22) International Filing Date: 15 January 1986 (15.01.86) (31) Priority Application Numbers: 692,711 805,504 803,452 (32) Priority Dates: (33) Priority Country: (71) Applicant: M-IE*-I 18 January 1985 (18.01.85) 29 November 1985 (29.11.85) 29 November 1985 (29.11.85) us A:PP:N fT fr Q I If fAPT 1-AIT (81) Designated States: AT (European patent), AU, BE (European patent), CR (European patent), DE (European pato.nt), FR (European patent), GB (European patent), IF (European patent), JP, LU (European patent), NL (European patent), SE (European patent).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

This docurni nt contains the f C' [tin dnents mlade unckr '3tf S,.to 49 and is correct for printing.______ 1 SEP 1986

AUSTRALIAN

13 AUG 1986 PATENT OFFICE B EseffR* 2'KER--HFT-bNf-DEt&E]; 2 5 0 Frankfttrter-Strasser-D-6-100--Darmstadt (DE).

(72) Inventor: HAHN, Gary, Scott ;7865 Camno~ Au'it, San Diego, CA 921921 (US).

(54) Title: IMM UNOREG ULATORY PEPTIDES Abstract NEUROLOIC CEFICII POINTS OF SJL/J MICE 1401 WIZ it SPINAL CORD 1IOONAIE IN CCoeLEC FRELIVS Peptides and peptide deriva- AJVfN RAE IE E EKW11SJ1fO f ETD Y-H tives, and method of using the same in muritmalian immune systems to 2.5. 0-0 SALINE suppress3 autoimmune responses, 0- LYS-N11 2 organ tiansplantation rejection re- 4Omtnoes injection or sponesor nopl~ticcel groth.saline or peptide dr nepSsipclirot.al5010 Cord In adjuvnart The peptides are characterized by the tx II11unization formula A-X-(B-Y)n-C wherein X and Y are residues of amino acids or0 amino acid derivatives with positive- 1.5 0 yI ciiarged side chains, Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His, or D-enantiomers of any of 1 these residues, A and C are- any substituents that preserve or augment OZ the immunoregulatory activity of the peptides, B is a residue of an, amino W 0./ acid or amino acid derivative that 0 preserves or augments the immu- 0 noregulatory activity of the peptide, 0 0 i and n isO0 or 1. The activity of Lite 10 1 2 5 0 5 1 subject peptides includes suppres. 4b1 23 2 3 4 sion of the proliferation of T-lymphocytes in in vitro systems that are DAYS r135I If.tIATIMp asnalogous to mammalian in vivo dis- )Nase conditions, regulation of tumor cell proliferation in vitro and in vivo, and reduction of autoimmune disease-associated isions in vivo. The peptides have potential human therapeutic benefits related to the treatment of autoimmune, orgyan or Ift rejection, neoplastic and other diseases, W 86/04334 PCT/EP86/00012 IMMUNOREGULATORY PEPTIDES BACKGROUND OF THE INVENTION The mammalian immune system consists of a collection of cells and molecules which contribute to the defense of the host against foreign and other undesirable agents including infectious agents and cancer. The molecular portion of the immune system *includes immunoglobulins, complement, histocompatibility antigens, and a variety of chemicals and peptide hormones which may either stimulate or inhibit the growth, development and activation of portions of the ce.lular immune system.

The cellular immune system consists of white blood cells (leukocytes) and includes lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils and mast cells, among others.

Depending on the cell type, leukocytes may circulate in blood or other body fluids, or may be fixed in various lymphoid organs including the thymus, bone marrow, lymph nodes and other organs.

In order to provide an effective immune defense, the cellular and molecular immune systems must interact in a highly intricate and complex manner. Optimally, the immune defense response should be precisely balanced to provide sufficent destructive force to eliminate infectious or eoreign agents or cancer cells without unnecessarily destroying healthy cells. A suboptimal immune response may result in uncontrolled growth of an infectious agent or cancer which may lead to the death of the 4 h WO 86/043 PCT/EP86/00 0 12 J WO 86/04334 2 host. A excessive immune response, by contrast, may destroy not only the intended target, but substantial quantities of surround- ing healthy tissue as well. The many molecules of the molecular immune system play a major role in regulating and "fine tuning" the cellular immune response to maximize destruction of infectious agents and cancer cells while minimizing destruction of surrounding healthy cells.

There are many diseases which are believed to occur because of a regulatory imbalance in the immune system. Autoimmune diseases are an example of conditions in which a substantial portion of an immune response is directed toward healthy host cells.

The causes for such misdirection of immune responsiveness are unknown for many diseases. Under normal conditions, the imlune system exhibits tolerance toward cells of the host which prevents the immune system from attacking normal, healthy cells.

It is this critical ability of the immune system to distinguish "self" from foreign cells and molecules that provides selectivity of an immune system attack. In autoimmune diseases, tolerance for host cells and molecules is reduced or eliminated resulting in significant destruction of otherwise healthy cells and organs.

The tendency of a host's immune system to display reduced tolerance to normal cells is strongly influenced by cell surface molecules whose genes are associated with the host's major histocompatibility complex (MHC). A particular MHC haplotype may substantially increase the risk of self-tolerance loss and subsequent autoimmunity. In certain autoimmune diseases, 1 'W86/04334 PCT/EP86/00012 infection by certain viruses or bacteria is believed to trigger the loss of self-tolerance which, in the setting of an appropriate MHC haplotype, results in an autoimmune disease. In other autoimmune diseases the triggering events which lead to a loss of self- toerance remain unknown.

Autoimmune diseases may affect every organ of the body.

Examples of diseases thought to have an autoimmune pathogenesis include, but are not limited to, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, Reiter's syndrome, Sjogren's syndrome, polymyositis-dermatomyositis, thrombocytopenic purpura, autoim.r'ne hemolytic anemia, ulcerative colitis, regional enteritis (Crohn's disease), chronic active hepatitis, primary biliary cirrhosis, idiopathic interstitial pulmonary fibrosis, Goodpasture's syndrome, postviral encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, juvenile onset insulindependent diabeteso Addison's disease, pernicious anemia, pemphigus, bullous pamphigoid and other diseases and conditions.

The immune response responsible for the rejection of transplanted organs among genetically non-identical animals or humans in many ways resembles an autoimmune disease in that an otherwise healthy transplanted organ may be destroyed by the 86/0433 PCTEP86/00012 SW O86/043 PCT/EP86/00012 WO 86/04334 PCT/EP86/00012 1 4 recipient's immune system. Such destruction occurs because the recipient's immune system recognizes the "foreign" histocomj patibility antigens present on cells of the transplanted organ and trigger a destructive immune response.

The complex regulation of immune responsiveness results from interactions between all classes of leukocytes, molecules secreted by leukocytes and cells and molecules from other organ systems. One class of leukocytes in particular, termed thymusderived lymphocytes or T-lymphocytes (T cells) is considered to be critically important to the coordination and regulation of imost immune responses. T cells may be divided into various subsets which have distinct immune functions. Helper T cells, for example, are critical for the growth and development of B cells into antibody-secreting plasma cells. 'Helper T cells are also critical for the growth and development of other T cell subsets such as Killer T cells which can directly destroy infectious agents, cancer cells, transplanted organs and, in autoimmune disease, healthy cells. By contrast, other T cell subsets termed suppressor T cells actively suppress the growth and development of B cells, Killer T cells and other lymphoid cells. Suppressor T cells are also critical for the development and maintenance of immune tolerance that prevents the development of autoimmune disease and organ transplantation rejection.

Because T cells have a powerful influence on the induction or suppression of the immune responses that lead to autoimmunity and organ transplantation rejection, pharmacologic agents whi(h regulate T cell functions may provide signific it therapeutic benefit in the treatment of human or animal disease.

i_ _1 0 'W 86/04334 PCT/EP86/00012 Thymulin and Thymic Hormone-Related Peptides Researchers have reported peptides that exhibit effects on the immune system, including effects relating to T-lymphocyte differentiation. In 1976, Bach et al. isolated and identified from porcine thymus the nonapeptide Serum Thymic Factor (FTS), now known as thymulin, having the sequence Glx-Ala-Lys-Ser-Gln- Gly-Gly-Ser-Asn (where "Glx" is either glutamine or pyroglutamic acid). (Bach, C. R. Acad. Sc. Paris, t. 283 (Nov. 29, 1976), Series D-1605; Nature 266:55 (March 3, 1977).) Thymulin has been shown to have hormonal properties related to the promotion of Tlymphocyte differentiation which include the induction of various antigenic markers on murine and human T-lymphocyte precursors, the'delaying of allogeneic skin graft rejection' in mice, enhancement of the generation of alloantigen reactive cytotoxic Tlymphocytes in thymectomized mice, modulation of thymic natural killer (NK) cell activity in tumor-bearing and NZB mice, inhibition of antibody-bearing lymphocyte production at late stages of differentiation, and suppression of Experimental Allergic Encephalomyelitis in guniea pigs. (Kaiserlian et al., Cellular Immunology 64:93 (1981); Kaiserlian et al., Cellular Immunology 66:360 (1982); Bardos et al., Clin. Immun. and Immunopath. 23:570 (1982); Kaiserlian et al., Clin. Immun. and Immunopath. 28:192 (1983); Immunology Today 4:16 (1983); Lenfant et al., Immunology 48:635 (1983); Nagai et al., J. Exp. Med. (Japan) 52(4):213 (1982).) Goldstein et al. have reported in U.S. Patent Nos.

4,215,112 and 4,232,008 that both T-lymphocyte and B-lymphocyte differentiation, as measured by antigen marker induction, may be induced with tripeptides and longer peptides containing the WO86/04334 PCT/EP86/00012 WO 86/04334 PCT/EP86/00012 6 sequence Lys-X-Gln, where X is chosen from specified amino acid residues. The sequence of these peptides resembles that of part of the thymulin molecule, but no determination of the mechanism of activity of the peptides is stated. Imaizumi et al. concluded in 1981 that the pentapeptide portion Lys-Ser-Gln-Gly-Gly is the minimum essential part of the thymulin molecule which retains full theta-antiger induction capability for T-lymphocyte precursors. (Imaizumi et al., FEBS Letters, 128:108 (1981).) Later studies found that other thymulin segments, namely Glu-Ala-Lys- Ser-Gln and Glu-Ala-Lys-Ser, act as antagonists to thymulin activity. (Gyotoku et al., Int. J. Peptide Protein Res. 21:135 (1983).) Goldstein et al. have described polypept.ides which mimic the ability of the thymic hormones thymopoeitin and splenin to induce the differentiation of T-'lymphocytes and/or B-lymphocytes.

U.S. Patent No. 4,190,646 discloses the pentapeptide Arg-Lys-Asp- Val-Tyr and certain derivatives capable of inducing the differentiation of bone marrow cells to T-lymphocytes. The dipeptide Gln-Lys and certain derivatives thereof are reported in U.S.

Patent No. 4,215,111 to be capable of inducing differenttiation of both T- and B-lymphocytes. Other pentapeptides capable of inducing T- and/or B-lymphocyte differentiaticln are described in U.S.

Patent Nos. 4,261,886 and 4,505,853.

A family of tetra- and pentapeptides which promote the differentiation of T-lymphocytes was described by Konig et al. in U.S. Patent No. 4,487,764. These peptides are composed of basic and aromatic amino acids in the sequence Basic-Basic-Aromatic- Aromatic-Optional.

d 86/04334 PCT/EP86/00912 7 Tuftsin and Related Peptides The tetrapeptide tuftsin, isolated by Najjar and described in U.S. Patent No. 3,778,426, has the sequence Thr-Lys-Pro- Arg. Tuftsin has been shown to stimulate in vitro phagocytosis by granulocytes, monocytes and macrophages. Other studies have shown tuftsin to be active in nanomolar concentrations in many species including humans, cows, dogs, rabbits, guinea-pigs and mice. In addition to its phagocytosis stimulating properties, tuftsin has been shown to stimulate such immune response functions as antibody-dependent cell-mediated cytotoxiclty (ADCC) and Natural Killer cell activity in mouse splenic cells, anti-bacterial activity of PMN-leukocytes and tissue macrophages, antigenspecific macrophage-dependent T-lymphocyte education and antibody synthesis to T-lymphocyte-dependent and independent antigens in, vivo and in vitro. Fridkin et al., Molecular and Cellular Biochemistry 41:73 (1981); Najjar et al., in Pick Lymphokine Reports (Academic Press 1980), p. 157; Nishioka, Life Sciences 28:1081 (1981). A number of active analogs of tuftsin have been identified, as well as inhibitors for tuftsin such as Lys-Pro- Arg, Ala-Lys-Pro-Arg and Ser-Lys-Pro-Arg. Peptide analogs which inhi/it the immunostimulatory activity of tuftsin do not, however, reduce the basal phagocytic activity of the cells.

(Najjar, Annals New York Academy of Sciences (198Z), p. 1; Najjar et in Pick Lymphokine Reports (Academic Press 1980), p. 157.) This suggests that immunosuppression could only be achieved via a mechanism distinct from that associated with tuftsin or its structural analogs.

Rigin, a tetrapeptide analog of tuftein having the peptide sequence Gly-Gln-Pro-Arg, has been shown to have i

J

WO 86/04334 PCT/EP86/0012 8 phagocytosis-stimulating properties similar to those of tuftsin.

(Veretennikva et al., Int. J. Peptide Protein Res., 17:430 (1981); U.S. Patent No. 4,353,823.) SUMMARY OF THE INVENTION It has been discovered that a number of very short peptides and peptide derivatives, including certain single-aminoacid derivatives, have a surprising ability to regulate T cell functions, and in particular to suppress T cell proliferation, and provide thereby potentially significant therapeutic benefits in the treatment of human or animal disease. Moreover, the peptides are capable of suppressing the growth of various neoplastic cells. Due to their short length, these peptides are relatively easy and inexpensive to manufacture. In addition, they are effective at non-toxic pharmacological dosage levels.

Accordingly, it is one object of the present invention to provide new peptides with important biological and pharmacological activities, as well as related and novel methods for using these peptjdes in suppressing the mammalian immune system in vitro and in vivo. A further object of the invention is to provide new peptides and methods related to the therapeutic I treatment of mammalian autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, and other diseases.

A further object of the invention is to provide new peptides and methods related to the therapeutic treatment of diseases involving a deficient, excessive or otherwise malfunctioning mammalian immune system response, including such conditions as organ transplantation rejection and bone marrow transplantation rejection.

r SW 86/04334 PCT/EP86/00012 9 A still further object of the invention is to provide new peptides and methods related to the treatment of neoplastic diseases.

Other objects and advantages of the invention will become apparent from an examination of the present disclosure.

The peptides of the present invention are characterized by the formula I -C I n wherein X and Y are residues of amino acids or amino acid derivatives with positively charged side chainsipreferably lysine, ornithin'e, arginine, or histidine, or D-enantiomers of any of these residues; A and C are any substituents, including amino acid residues, that serve to preserve or augment the immunoregulatory activity of the peptide; B is a residue of an amino acid or amino acid derivative that preserves or augmento the immunoregulatory activity of the peptide; and n is 0 or 1.

Thus, the peptides of the present inventin can be characterized either by formula A-X-C (la) or by formula A-X-B-Y-C Compounds of formula Ia are preferred.

Most preferably, in Ia, X will be chosen from among the amino acids lysine, ornithine, D-lysine and D-ornithine; in Ib, X will be most preferably lysine, arginine, histidine or ornithine, while Y will most preferably be lysine, arginine or his idine.

L

WO 86/04334 PCT/EP86/00012 A may be, for example, hydrogen or an N-acetyl, N-acyl, N-aliphatic, or N-aromatic group; or A may be an amino acid such as alanine, glycine, leucine, valine, phenylalanine, tyrosine, asparagine, aspartic acid, sarcosine, glutamine, glutamic acid, serine, or threonine, or an amino acid derivative including D-enantiomers of these amino acids, such as D-alanine or D-aspartic acid.

B may be, for example, a residue of an amino acid such as valine, leucine, glutamic acid, glutamine, serine, threonine, glycine, alanine, proline, asparagine, aspartic acid, arginine, histidine, isoleucine, tyrosine, phenylalanine, lysine, ornithine, or tryptophan,, or of a D-enantiomer or dther derivative of an amino acid, such as D-valine or D-serine.

C preferably will be a small uncharged chemical constituent, for example -NH 2 -NHR or -OR, where R is .preferably H or a lower alkyl; or C may be a residue of an amino acid such as glycine, alanine, leucine, valine, serine, cysteine, sarcosine, threonine, proline, glutamine, phenylalanine, tyrosine, homoserine or a D-enantiomer or other derivative of an amino acid.

It wil be apparent from the following disclosure that significant immunoregulatory activity will be achieved for a large nrumber of diverse substituents in the A, B and C positions, so long as the constituents X and Y are as dofined above. Thus, in Ib, (W 86/04334 PCT/EP86/000 12 imrunoregulatory activity may be retained wjhere either or both of the terminai grb(Jps A and C comprise multiple ifio acids that do not interfer'e substantially with the immunoregulatory activity associated w-1th the central tripeptide sequence X-B-Y. Accordingly, the present invention specifically contemplates that derivatives, inc1,uding homologs, analogs, enantiomers, extensions, or other substitutions not specifi~cally disclosed herein are within the scope and~ spiric of the present invention.

DETAILED DESCRIPTION OF THE INVENTION In. the -following discussion, the amino acixd components., of immunoregulatory peptides are frequently identified with abbreviations for convenience. Unless otherwise specified, th'e foll.owing abbreviations designate L-enantiomeric forms of the amino acids, as follows: Amino Acid Abbreviation glycine Gly L-alani ne Ala L-valine Val1 L-leucine Leu L-,isoleucine Ile L-proline Pro L-methionine Met L-cysteine Cys L-phenylalanine Phe L-tyrosine Tyr PCT/E P86/000 12 )~7 4 1 1 WO $6/04334 In substitutes L-tryptophan Trp L-histidine His L-lysine Lys L-arginine Arg L-aspartic acid Asp L-asparagine Asn L-qlutamic acid Glu L-glutamine Gln L-serine Ser L-threonine Thr L-ornithine Orri sarcosine Sac L-homoserirne HomoSer addition, the following abbreviat~ions for chemical are used: Substituent N C, -substituted amino acid N -acylated atzino acid N -acetyl, amino acid N -rnethyl amino acid des- 4 -ernino acid Amino~ acid amide Abbreviation N-R-(amino acid) RCO-(amino acid) Ac-(amino acid) N-Me-(amino acid) desamino-(amino acid) (Amino acid)-NH 2 -NHR,

-NR,

(Amino acid)-OR.

Amino acid ester 86 4 PCT/EP86/00012 SWO 86/04334 13 As used herein, the term, "amino acid derivative" refers to homologs, analogs, D-enantiomers, and chemically substituted or otherwise modified forms of amino acids such as those listed above. As will be seen from the following description of the invention, it has been shown that a wide range of derivatives may be substituted for naturally-occurring amino acids in the peptides of the present invention while still preserving or augmenting immunoregulatory activity. For example, chemical substitution of acyl, alkyl, and other substituents at the N-terminus, or formation of esters or amides at the C-terminus, will frequently yield peptide derivatives with preserved or augmented immunoregulatory activity.

Furthermore, the present invention contemplates that certain amino acijl within the disclosed peptides may be substituted by amino acids which are chemically similar by virtue of similar side chain size, charge, .shape, solubility, or other chemical characteristics while still retaining the peptide's immunoregulatory activity. Amino acids with such chemical similarity are termed "functionally conserved." Functional classes of the common genetically-coded amino acids have been specified by Dayhoff, et al. in Atlas of Protein Sequence and Structure, volume 5 (National Biomedical Research Foundation, page 98. Derivatives of the naturally-occurring amino acids, such as ornithine, homoserine, homolysine, des-N-amino lysine and homoarginine, have chemical structures and properties comparable to their naturally-occurring analogs or homologs, and PCT/EP86/00012 WO 86/04334 14 thus may, in proper cases, be substituted in the peptides of the present invention to preserve or even increase immunoregulatory activity.

One aspect of the immunoregulatory activity of the subject peptides resides in the capability of the peptides to suppress autoimmune responses, to suppress organ transplantation rejection responses, or to suppress neoplastic cell growth. As will be discussed below, such immunoregulatory activity may be realized in in vitro systems that are modeled after in vivo mammalian disease conditions, as well as in in vivo systems.

The £mmunoregulatory peptides of the present invention are characterized by the formula -C I n wherRin X and Y are residues of amino acids or amino acid derivatives with positively charged side chains at physiological pH pH 6 to 8) or D-enantiomers of any of these residues; A and C are amino acid residues or other substituents, that preserve or augment the immunoregulatory activity of the peptide; B is a residue of an amino acid or amino acid derivative that preserves or augments the immunoregulatory activity of the peptide; and n is 0 or 1.

S'Wb86/04334 PCT/EP86/00012 Thus, in Ia, X may be chosen from among residues of such naturally-occurring positively charged amino acids as Lys, Arg and His.

In addition, however, it has been discovered that residues of positively charged amino acids other than those naturally-occurring acids listed above may be used successfully in the X position. For example, substitution of the D-enantiomeric form of the amino acid X will, in some cases, augment, and, in many cases, substantially preserve the immunoregulatory activity of the subject peptide. As another example, residues of desamino forms of the central amino acid X, wherein the N -amino group of X is absent, may preserve or augment the immunoregulatory activity of the peptide.

Other amino acid derivatives, including homologs, analogs, enantiomers and otherwise modified forms of naturally occurring positively charged amino acids, can also be expected to yield significant immunoregulatory activity. As an example, ornithine, which is closely homologous to lysine, demonstrates significant activity despite the fact the ornithine does not occur naturally in proteins or in other molecular constituents of the immune system.

Especially preferred fot X are Lys and Orn. The D-enantiomeric form of these amino acids (D-Lys, D-Orn) as well as the deaminated forms (desamino-Lys, desaminotOrn) may be substituted for the naturally occurring L-enantiomer WO 86/04334 PCT/EP86/00012 16 in proper cases, with the result that immunoregulatory activity or the peptide is substantially preserved or augmented.

In Ib, the structure of the three central constituents of the subject polypeptides is of central importance. In its simplest from, Ib countenances a tripeptide of the form

X-B-Y

wherein X and Y are any positively-charged amino acids separated by any single amino acid, B, which tripeptide has immunoregulatory activity. In this form of the present invention, terminal constituent A is hydrogen and terminal constituent C is a hydroxyl group. X and Y may be the same or different positively charged amino acids, preferably chosen from among Lys, Arg, His and Orn.

Lys and Arg are particularly preferred in the X and Y positions. Thus, the following tripeptide sequences are particularly preferred in the present invention: Lys-B-Lys Arg-B-Arg Lys-B-Arg Arg-B-Lys In addition to the tripeptide sequences given above, immunoregulatory activity may be achieved through the use of 1 W0'86/04334 PCT/EP86/00012 17 His or Orn in the X or Y positions. His or Orn may be substituted in the X position, while His is preferred in the Y position.

In addition to the common forms of the amino acids discussed above, the present invention contemplates that modified, homologous, analogous or artificial forms of positively charged amino acids may be utilized in the X and Y positions of Ib. Thus, D-enantiomeric forms of positively charged amino acids may, in appropriate combination with other constituents of the subject polypeptides, substantially preserve or augment the immunoregulatory activity of the peptide.

Constituent B comprises the residue of any amino-acid or amino acid derivative which preserves' or augments the immunoregulatory activity of the subject polypeptides. It has been discovered that a very broad range of amino acids may be incorporated into the B positic o t.o yield polypeptides with significant immunoregulatory activity. Preferably, B will be chosen from among the residues of the following amino acids or their D-enantiomers: Val, Lu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Trp.

Most preferably, B will be chosen from among the following amino acids or their D-enantiomers: Val, Leu, Glu, Gln, Ser, Thr, Gly, Ala.

;1 WO 86/04334 PCT/EP86/00012 18 As discussed above, the B position amino acids listed are preferably used in conjunction with the positively charged amino acids Lys or Arg in the X und Y positions.

In peptides in which both X and Y are Lys, the amino acids Val, Glu and Gin are especially preferred in the B position.

When both X and Y are Arg, Asp and Ser are highly active substituents in the B position. Ser and Thr are examples of suitable B substituents when X is Lys and Y is Arg, while Gly, Ala, Ser and Thr are all active when X is Arg an'd Y is Lys.

The terminal constituent A, which is substituted at the N -position of X, may be chosen from a number of chemical groups or substituents. For example, terminal constituent A may be hydrogen alone, yielding a peptide of the general formula -C.

As another example, constituent A may be an N-acetyl substituent, thus yielding a peptide of the general formula Ac-X-(B-Y) -C.

Other substituents in the A position that may preserve or augment immunoregulatory activity include aliphatic and aromatic acyl substituents of the form RCO-, as well as substituents of the form where R is preferably an unbranched or. branched alkyl group of one to eight carbons, and may also be C 2

-C

8 alkenyl, C 2

-C

8 alkynyl,

C

6

-C

1 4 aryl, C 7

-C

1 4 alkaryl, C 7 Cl aralkyl, or C 3 -C14 cycloalkyl.

SWO'86/04334 PCT/EP86/00012 19 Furthermore, constituent A may be an amino acid.

Ala and 0-Ala are especially preferred amino acids for constituent A. Also preferred as amino acids for terminal constituent A are the following: Gly, Leu, Val, Phe, Tyr, Asn, Asp, Sar, Gin, Glu, Ser and Thr as well as the D-enantiomers of these amino acids.

In addition to'single amino acid substitutions in the A terminal position, substitutions of more than one amino acid have been shown to yield active polypeptides, particularly of formula Ib. In particular, A terminal constituents of the form Val-Asp-, Gly-Asp-, Ala-Asp-, Ser-Asp-, Thr-Val-Asp- and Leu-Thr-'Val-Asp- have been successfully used in the A position. Thus, it is believed that the immunoregulatory activity of the present polypeptides may be preserved with a large number of diverse substituents, including sequences containing multiple amino acids, in the A position as well as in the C terminal position.

It will be apparent to those skilled in the art that modifications, additions, deletions, or substitution to the chemical groups named above as A terminal constituents may preserve activity without departing from the spirit of the invention described herein.

Terminal constituent C also may be chosen from a number of chemical groups or substituents which preserve or augment the immunoregulatory activity of the subject

I

I I 2 WO 86/04334 PCT/EP6/00012 peptides. In general, it is preferred that C be a relatively small, uncharged species (including a hydroxyl-(-OH) group Furthermore, amidation, esterification and addition of one or more amino acids is possible in the terminal group C. For example, one preferred embodiment of C is of the form

-NHR

wheiein R is hydrogen or a lower alkyl, such as C 1 -QC alkyl substituent. As an example of the latter, wherein R is ethyl, and where X is Lys and A is hydrogen, Lys-NHCH 2

CH

3 is representative.

Other substituents in the C terminal position may preserve or augment immunoregulatory activity, most preferably relatively small and uncharged species. Thus, R groups such as

C

2

-C

8 alkenyl, C 2 -Cg alkynyl, C 6

-C

14 aryl, C 7

-C

1 4 alkaryl, C 7 -C14 aralkyl and C 3

-C

14 cycloalkyl may be utilized in C terminal substituents of the form -NHR or -OR to yield amides and esters, respectively. Secondary amino groups of the form -NR 2 may also be expected to yield active peptide amides.

Particulaily significant immunoregulatory activity may be obtained by using lower alkyl R groups in the polypeptide amides described above. C 1

-C

3 lower alkyl groups are particularly preferred. In particular, significant activity has been obtained in tripeptide amides of the form Lys-B-Lys-NH 2 WO 86/04334 PCT/EP86/00012 21 wherein B is Val, Leu, Pro, Glu, Gln or Gly.

Contrarily, amidation where the B position amino acid is Asp, Ala, T' Arg, or His yields peptides with lower, altho., .till significant, immunoregulatory activity.

Amino acids and derivatives thereof may also be used as C terminal constituents. In general, it is particularly preferred that such amino acids have relatively small, uncharged side chains. Thus, Ser, Gly, Ala, Leu, Val and Gys, are particularly preferred as C terminal constituents.

Ser has been shown to yield a particularly high immunoregulatory activity, particularly in the combination Lys-Ser. Gly and Ala are especially preferred in the combinations Orn-Gly, Lys-Gly and Orn-Ala.

As with the examples discussed above with respect to amino acid constituents at the A and X positions, the D-enantiomeric form of amino acids in the C cerminal position may demonstrate significant immunoregulatory activity.

The combination Orn-D-Ala is especially preferred.

Although amino acids having relatively small, uncharged side chains are particularly preferred as C constituents in the present invention, significant immunoregulatory activity is also achieved using amino acids with relatively larger uncharged side chains as C terminal constituents. Also prefered, therefore, as C terminal constituents are the following amino acids, as well as their D-enantiomers: Thr, Pro, Gin, Phe, Tyr.

I- WO 86/04334 PCT/EP86/00012

I

22 Other amino acids, or derivatives thereof, including homologs, analogs, enantiomers, or combinations thereof, as well as other non-amino acid chemical constituents, may likewise be incorporated into the C terminal position to substantially preserve or augment the immunoregulatory activity of the subject peptides. For example, HomoSer and Sar respectively, may be utilized successfully in the present invention as C terminal constituents.

In addition, amides or esters of C terminal amino acids may be so used. The peptide Lys-Ser-NH 2 is particularly preferred. Furthermore, combinations or sequences of amino acids which substantially preserve or augment the imunoregulatory activity of the subject peptides may be incorporated as C terminal constituents. It is believed that it is within the skill of the applicable art to identify and utilize as C terminal constituents those chemical substituents, including amino acids and their derivatives, that substantially preserve or augment the inmmunoregulatory activity of the subject peptides, and which are not disclosed specifically irein.

It is apparent from the foregoing discussion that a peptide of the present invention may incorporate one, two, three, or more amino acids. In its simplest form, the present invention contemplates a single, positively charged amino acid (the central amino acid, X) flanked by non-amino acid terminal constituents A 0'86/04334 PCT/EP86/00012 23 and C. Certain of these "monopeptides" demonstrate extremely high immunoregulatory activity. Particularly preferred are lower alkyl amides of Lys, Orn, D-Lys and D-Orn.

Thus, the following monopreptides are particularly preferred: Lys-NH2 Lys-NHCH Lys-NHCH 2

CH

3 D-Lys-NE 2 D-Lys-NHCH 3 D-Lys-NH 2

CH

3 Orn-NH 2 Orn-NHCH 3 Orn-NHCH 2

CH

3 D-Or n-NH 2 D-Orn-NiHCH 3 D-Orn-NHCH 2

CH

3 The present invention countenances a large number of highly active dipeptides. Many of these are characterized by an A terminal constituent that comprises a non-amino acid species and a C terminal constituent that comprises an amino acid or a homolog, analog, or other derivative thereof. In particular, it is particularly preferred that the C terminal constituent be Ser, Lys, A1 or Cys, and that the central amino acid X be Lys or Orn. The A terminal constituent is preferably hydrogen oi an acetyl group.

WO) 86/04334 P(T/EP86/00012 24 In addition, the C terminal constituent may be amidated to form a lower alkyl amide. Thus, dipeptides of the following general form are particularly preferred: X-C

X-C-NH

2

X-C-NHCH

3 X-C-NHCH 2 CH 3 Apc-X-C Ac->X-C-NH 2 Ac-X-C-NHCH 3 Ac-X-C-,NHCH 2

CH

3 wherein C is preferably Ser, Glyp Ala, or Cys, and X is preferably Lys or Orn. The combinatilons, Lys-Ser, Lys-Gly, Lys-Ser-NH 2 Orn-Gly and Orn-Ala are especially The use of D-enantiomeric forms of amino acids in the! X and C positions has been shown to substantially preserve or augment immunoregilatory activity in a number of cases.

Particularly preferzed D-enantiomeric substitutions inc'ude, in the central amino acid X position, D-lysine and D-ornithine, and, in the C terminal position, D-alanine. D-Lys-Ser and Orn-D-Ala have been showai t.,o have particularly high immunoregulatory activity.

W PCT/EP86/00012 WO 86/04334 In addition to the foregoing class of dipeptides, in which the A terminal constituent is not an 'mino acid, the present invention contemplates that class of dipeptides characterized by an amino acid A terminal constituent in conjunction with a non-amino acid C terminal constituent. In general, for la,it is preferred that the C terminal constituent be a group other than a hydroxyl group although dipeptides such as Asp-Lys and Ser-Lys, in which Cis a hydroxyl group, have been shown to exhibit immunoregulatory activity. A C terminal substituent of the form -NHR, wherein R is preferably hydrogen or a lower alkyl, accompanied by an A-position amino acid as discussed below, will yield a dipeptide amide of the central positively charged amino acid X. Amides of th~s central amino acid have been shown to have particularly significant immunoregulatory activity.

Particularly preferred as A terminal amino acids are Ala, Gly, Val, Phe, Tyr, Asp and Sar, as well as the ;nantiomers of these anino acids. Ala is especially preferred as an A terminal constituent in peptides of the form Ala-X-C wherein X is preferably Lys, D-Lys, Orn, or D-Orn, and C is preferably Gly, Ala, D-Ala, or Ser. In addition the use of D-Ala as an A terminal constituent is preferred, yielding, f.e. a peptide of the form D-Ala-X-C, 27 wherein X and C are preferably as defined immediately above.

B

WO 86/04334 PCT/EP86/00012 26 Modifications of the C terminal constituent, as for example, by amidation to a lower alkyl amide, may also yield peptides having potent immunoregulating activity. Other modifications to the constitusnts of the subject peptides will be recognized by those skilled in the art and can readily be synthesized, tested and utilized within the scope of the present invention as described herein.

Specifically preferred are peptides of formulae laa to lam, lama to Iamz and Ian to lat which correspond to formula Ia, but wherein in Iaa: X is a residue of an amino acid or amino acid derivative with a positively charged side chain; A is H, R, RCO, or a residue of an amino acid or amino acid derivative; C is NH 2 NHR, NR 2 OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C 1

-C

8 aliphatic, C 6

-C

1 4 9rvl, aralkyl or alkaryl, or C3-C14 cycloalkyl; in lab: X is a residue of an amino acid or amino acid derivative with a positively charged side chain; A is H acetyl, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr; and WdO86/04334 PCT/EP86/00012 27 C is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; in Tac: X is a residue of an amino acid or amino acid derivative with a positively charged side chain; A is any chemical substituent, residue or an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; and C is NH 2 NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-G3in, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester,formed by substitution of one of the foregoing amino acids with a substituent of the form

NH

2 NHR or OR, wheqre R is a C 1

-C

8 alkyl; in lad: X is a residue of an amino acid or amino acid derivative with a positively charged side chain; A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, 0-Phe, 0-Tyr, D-Asp, D-Ser or D-Thr; C is NH 2 NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSor) D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, 0-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or est er formed by substitution of one of the foregoing amino arids with a substitent of t'e form NH 2 NHR, or OR; and R it a C 1 CB alkyl; W'd 86/0433 PCT/EP86/0)012 WO 86/04334 PCT/EP86/00012 28 in Iae: X is a residue of an amino acid or amino acid derivative with a positively charged side chain; A is H, acetyl, Ala, and D-Ala; and C is NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is a Cl-C alkyl; in laf: X is Lys Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His; A is any chemical -substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; and C is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; In lag: X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Art or D-His; A is H, R, RCO, a residue of an amino acid or an amino acid derivative; I C is NH 2 NHR, NR 2 -OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a 3ubstituent of the form NH 2 NHR, NR 2 or OR; and R is a C -C 8 aliphatic, C -C 1 4 aryl, aralkyl or alkaryl, or C3-C14 cycloalkyl; I I t WO 86/04334 WO'8604334PCT/EP86/00012 in lah: X is Lys, Orn, Arg, His, 0-Lys, 0-Orn, D-Arg, or 0-His; A is H, R, RCO, Ala, Gly, VSal, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, 0-Val, 0-Phe, 0-Tyr, D-Asp, 0-Ser and D-Thr; C is NH NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSer, D-Gly, 0-Ala, 0-Ser, D-Cys, D-Ser, D-Thr, 0-Pro, D-Gln, D-Phe, 0-Tyr, 0-HomoSer, or a residue of an amino acid amide and or Pestgr formed by substitution of one of the foregoing amino acids with a substituent of the form NH2, NHR, or OR; and *R is a C I- C 8 lower alkyl; in Iai: or 0.-His; X is Lys,, Orn, Arg, His, 0-Lys, 0-Orn, D-Arg, A is H, acetyl, Ala, or 0-Ala; and C is NHR', Gly, Ala, Sei, 0-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is a C 1

C

8 alkyl; in Iaj: X is Lys, Orr., 0-Lys or 0-Orn; A is pn~ chemical substituent, residue of an amino acid, or amino acid derivative that preserves the immunoregulatory activity of said peptidE,; and C is any chemical substituent, residue of an amino acid, or amino acid derivative that preserves the immunoregulatory activity of said peptide; I L. Imywa y ue acnievec tnrougn me use or WO 86/04334 PCT/EP86/00012 in lak: X is Lys, Orn, D-Lys or D-Orn; A is H, R, RCO, or a residue of an amino acid or amino acid derivative; C is NH 2 NHR, NR 2 OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C 1 -C aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or C3-C14 cycloalkyl; 3 14 in lal: X is Lys, Orn, D-Lys or D-Orn; A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr; and C is NH 2 NHR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gin, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent ol the form NH 2 NHR, or OR; and R is a C 1 -C alkyl; in lam: X is Lys, Orn, D-Lys of D-Orn; A is H, acetyl, Ala, or 0-Ala; and C is NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is

C

1

-C

8 alkyl; 7 i W'8~6/04334 PCT/EP86/OO 12 in Iama: Lys or D-Lys; H; and -NHR,where R is H, methyl or ethyl; in Iamb: in Iamc: in Iamd: in lame: Orn or D-Urn, H; and NHR, where R is H, methyl or ethyl; Lys H or acetyl; and Gly or Gly-NHR, where R is H, methyl or ethyl; Lys; Ala; and Gly or Gly-NHR, whe-re R is 1,methyl or ethyl; D Ly s H or acetyl; and Gly or Gly-NHR, where R is H, methyl or ethyl;, 0Urn H or acetyl; and Gly or Gly-NHR, where R is H, methyl or ethyl; D0Urn H or acetyl; and Gly or Gly-NHR, where R is H, methyl or ethyl;, Lys H or acetyl; and Ala or Ala-NHR, where R is H, methyl or ethyl; in Iamf: in Iamg: in Iamh: WO 86/04334 WO 8604334PCT/EP86/000 12 I

I

in lami D-Lys; H or acetyl; and Ala or Ala-NHR, where R is H, methyl or ethyl in Iamj: or ethyl; in Iamk: Lys H or' acetyl; and D-Ala or D-Ala-NHR, where R is H, methyl D-Lys; H or acetyl; and 0-Ala or D-.Ala-NHR, where R is H, methyl Orn; H or acetyl; and Ala or Ala-NHR, where R is H, methyl or or ethyl; in Iaml: ethyl,,, in Iamm: 0-Orn; H or acetyl; and Ala or Ala-NHR7 where R is H, methyl or e th yl1; in Iamn: 0 r; H or acetyl; and 0-Ala or D-Ala-NHR, where R Is H, methyl or ethyl; IVO'86/04334 WO'8604334PCT/EP86/00012 in Iamo: e t hyl1; in lamp: e t h yl; in Iamq: D-Orn H or acetyl; and D-Ala or D-NHR, where R is H, methyl or L ys H or acetyl; and Ser or Ser-NHR, where R is H,methyl or D-Lys H or acetyl; and Ser or Ser-NHR, where R is H, methyl or Lys; H or acetyl; and D-Ser or D-Ser-NHR, where R is H, methyl ethyl; in Iamr: or ethyl; in Iams: D-Lys H or acetyl; and D-Ser or D-Ser-NHR, where R is H, methyl or ethyl; in Iamt: Orn H or acetyl; and Ser or Ser-NHR, where R is H, methyl or ethyl; in Iamu: D-Orn;, H or acety; and Ser or Ser-NHR, where R is H; methyl or athyl; WO 86/0i334 PCT/EP86/OO 12 I I in Iav.: X

A

C

or ethyl; is Orn; is H or acetyl; and is D-Ser or D-Ser-NHR, where R is H, methyl in Iamw: is D-Orn; is H or acetyl; and is D-Ser or D-Ser-NI-R, where R is H, methyl or ethyl; in Iarmx: is Lys or is Ala or is Cly; is Lys or is Ala or is Ala or D Lys; D-Ala; and D-Lys; D-Ala; and D-*Ala in lamy: in Iamz: X is Lys or D-Lys; A is Ala or D-Ala; and C is Ser or D-Ser; in Ian.- X is a residue of an amino acid or amino acid derivativ~e with a positively charged side chain; A is Ala, V/al, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-.Tyr, D-Asp, D-Ser or D-~Thr; 0 it Cys, Sar, Thr, HomoSero D-Ala, D-Ser, D-Cys, D-Tt1f) D-PrPI, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by gubstitution of one of the foregoing gmino acids with a substituent of the form NH 2 NHR or OR; and V o '86/04334 PCT/EP86/ O00012 R is a C-C 8 aliphatic, C6-C1 aryl, aralkyl or alkaryl, or C3 C 4 cycloalkyl; in lao: X is Lys, Orn, Arg, His, D-Lys, 0-Orn, D-Arg, or D-His; A is Ala, VJal, Tyr, Asp, Sar, Ser, Thr, D-Ala, 0-Phe, D-Tyr, 0-Asp, 0-Ser or D-Thr; C is fys, Sar, Thr, HomoSer, or a residue of an a~mino acid amide or ester formed by substitution of one of the foregoing amino acids with a oiubstituent of the form NH 2 NHR or OR; and R is a C-C 8 aliphatic, %C C aryl, aralkyl 11 3 6114 or alkaryl, or C C cycloalkyl; 3 14 qin lap: X is Lys or 0-Lys; A is R, RCO, Ala, Gly, VIal, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-V~al, D-Phe, 0-Tyr, i-Asp, 0-Ser, or D-Thr; C is NH, 'NHR, OR, GJly, Ala) Ser, Cys, Sar, Thrt Pro, Gin, Phe, Tyr, k joSeg, 0-Ala, D-Ser, 0-Cys, D.-Thr, D-Pro, D-Gln, D-Phej D-Tyr, D-Homo~er, o~r a residue of an amino acid amide or ester formed by sub~stitution of one of the foregoing amino acids with a substituent of the form NH 2 NHR or OR; and R is a C 1 -C 8 Ioweir alkyl; in Iaq: X is Lys or 0-Lys; A is acetyl, Ala or 0-Ala; and WO 86/04334 WO/034PCT/EP86/00012 II 36 C is NH 2 NHR, Gly, Ala, Ser, 0-Ala, D-Ser, Gly-NHRI Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is a C 1

C

8 alkyl; in Iar: X is Orn or C-Orn; A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar Ser Thr D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr; C is NH 2 NHR, NR 2 OR, or a residue of an amino acid or amino acid amide or ester formed by substitution-of an amino acid with a substituent of the form NH 2 NHR, NR 2 or OR; R is a C 1

C

8 aliphatic, C 6

C

14 aryl, aralkyl or alkaryl, or C 3 -C 14cycloalkyl; provided that Orn-Ala and Orn-NH 2 are not included; in las: X is Orn or D-Orn; A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, D-Ala, D-Val, D-Phe, D-Tyr or D-Asp; and C is NH 2 NHRI Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gin, Phe, Tyr-, HomoSer, i)-Ala, D-Ser, D-Cys, D-Thr, 0-Pro, 0-Gin, D-Phe, D-Tyr, D-HomoSer, or a residue of an am~ino acid amide or ,-ster formed by substitution o i one of the foregoing aminn, ucids with, a substituent of' the d1.orm NH 2 NHR or OR; R is a C 1

C

8 alkyl; prov4ided that Orn-Ala and Orn-NH 2 are not included; IWO 86/04334 PCT/EP86/00012 37 in lat: X is Orn or D-Orn; A is H, acetyl, Ala or D-Ala; and C is NH 2 NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR or D-Ser-NHR, wehre R is a C 1 -Cg alkyl; provided that Orn-Ala and Orn-NH 2 are not included.

Specifically preferred are, furthermore, peptides of formulae Iba to Ibz and Ibza to Ibzo which correspond to formula Ib but wherein in Iba: X and Y are residues of amino acids or amino acid derivatives with positively charged side chains; A is H, R,.RCO, amino acids, and amino acid derivatives formed by N -substitution of an amino acid with a substituent of the form R or RCO; B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; C is OH, NH 2 NHR, NR,, OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C -C 8 aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or C 3

-C

1 4 cycloalkyl; in Ibb X and Y are residues of amino acids or amino acid derivatives with positively charged side chains; WO 86/04334 PCT/EP86/00012 38 A is H, R, RCO, or a residue of any amino acid or amino acid derivative that preserves the immnunoregulatory activity of said peptide; B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; C is OH, NH 2 NHR, NR 2 OR, or a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; and R is a -C aliphatic, C-C 1 4 aryl, araikyi or alkaryl, or C -C 1 4 cycloalkyl; in Ibc; X and Y are each residues of amino acids or amino acid derivatives with positively charged side chains A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp, D-Ala, D-Val, -Leu, D-Asn, D-Gloi, D-Ser, D-Thr, or a residue of a derivatie of one of the foregoing amino acids formed by N substitution with a substituent of the form R or RCO; B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lie, Tyr, Phe, Lys, Orri, Trp, or a residue of a D-enantiamer of one of the foregoing amino acids; C is OH, NH 2 NHR, NR 2 OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, O-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one %W0'86/04334 PCT/EP86/OO 12 39 of the foregoing amino acids with a substituent. of the form NH1 2 NHR, NR 2 or OR; and R is a C 1

C

8 aliphatic, C 6

C

1 4 aryl, aralkyl or alkaryl, or C 3-C 1 cycloalkyl; in Ibd: X and Y ai~e each residues of amino acids or amino acid derivatives with positively charged side c h ains; A is H, R, RCO, Asp, Gly, Ala, VJal, Leu, Asn, Glu, Ser, Thr, 0-Asp or a residue of a derivative of one of the foregoing amino acids formed N' substitution with ,o subs9tituent of the form R or RCO; B is VIal, Leu, Glu, G~n, Ser', Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Tt'p, D.-Val, D-Ser, or D-Ala! i, 5C is OH, N'H 2 NH1R, Gly, or Ser; and R is a C 1 C 8 alikyl; in Ibe: X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, 0-His, or D-Orn; A is a chemical substituent, residue of an amino 'it) acid, or amino acid derivative, or sequence of amino acids or amino acid derivati' that preserves the immunoregulatory activity of said peptide; B is a regidue of an amino acid or smino acid derivative thet preserveis the immunoregulatory (4,ivity of said peirtide; and C is a chemical sUbstif-uent, residue of an amino acid oi, amino acid derivative, or sequence of C,,ii.no WO 86/04334 PCT/EP86/00012 acids or amino acid derivatives, that preserves the j immunoregulatory activity of said peptide; k in Ibf- X an Yare each Lys, Arg, His, Orn, D-Lys, D-Arg, 0-His, or D-Din; A is H, R, RCO, or a residue of an amino qcid or amino acid derivative, forined by N0--substitution of an amino acid vit.h a substituent of the form R or RCO; B is a regiduo of an amino acid or amino acid derivative that preserves the immunorecgulatory activity of said peptide; C is OH, NH 2 NHRI NR, OR,or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of th~e form NH 2 NHR, NR, or OR; and R3 is a C-C 8 ali p h at ic, C 6 -C aryl, aralkyl or alkaryl, or C 3

-C

1 cycloalkyl; 3V1 in Ibg: X and Y are each Lys, Arg, His, Orn, D--Lys, D-Arg, 0--His, or 0-Oin; A is H, R3, RCO, or a residuea of amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide~ B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity Wj said peptide; C is OH, NH 2 NHR, NR 2 OR, or a residue of an anino acid or amino acid derivative that preserves the !mmunoreguiatoiny activity of said peptide; and IWO686/04334 PCT/EP86/0')0012 4 J.

R is a C-C 8 ai.iphatic, C C 1 aryl, aralkyl or alkaryl, or C -V cycloalkyl; 3 1.4 in Ibh: X and Y are each Lys, Arg, His, Orn, Ci-Lys, D-Arg, D-His, or D-Orn; A is H, R, RCO, Asp, Gly, Ala, VSal, Leu, Asn, Glu, Ser, Thr, D-Asp, D-Ala, D-\Ial, D-Leu, D-Asn, D-Glu, D-Ser, D-Thr, or a residue of a derivative of one of the foregoing amino acids formed by N -substitution wit-h a substituent of the form R or RCO; B is VSal,, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Urn, Trp, or a residue of one of-'the D-enantiomers of the fore- 1 going amino acids; OGy eAa Vl, euD-Sr, -Ala D-alD-Lu, r aresidue of a amno cidamie orestr frme bysubstitution of one of the foregoing amino acids with a substituent of the form~ NH, NHR, NR 2 or OR; and R is a C 1 -C a al iphatic, C 6-C "4ay aralkyl 120 or alkaryl, or C C 1 cycloalkyl; in Ibi: X and Y are each Lys,Arg, His, Urn, D-Lys, D-Arg, D-His, or D-Orn; A is H, R, RCO, Asp, Gly, Ala, \/al,Leu, Asn, Glu, Ser, Thr, D-Asp or a residue of a derivative of one of the foregoirj amino acids formed by N -subsLitution with a substituent of the form R or RCO; d WO 86/04334 PCT/EP86/00012 42 B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala; C is OH, NH 2 NHR, Gly or Ser; and R is a C 1

-C

8 alkyl; in Ibj: X and Y are each Lys, Arg, His, or Orn; A is a chemical substituent, a residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immun:cregulatory activity of said peptide; B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; and C is a chemical substituent, a residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immunoregulatory activity of said peptide; in Ibk: X and Y are each Lys, Arg, His, or Orn; A is H, R, RCO, or a residue of an amino acid or amino acid derivative formed by N -substitution of an amino acid with a substituent of the form R or RCO; B is a residue of an amino acid or amino acid derivative that preser'!es the immunoregulatory activity of said pept-de; C is OH, NH 2 NHR, NR 2 OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of 86/04334 PCT/EP86/00012 43 an amino acid with a substituent of the form NH 2

NHR,

NR

2 or OR; and R is a C 1

-C

8 aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or C3-C14 cycloalkyl; in Ibl: X and Y are each Lys, Arg, His, or Orn; A is hydrogen, R, RCO, or a residue of amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; C is OH, NH 2 NHR, NR2, OR, or a residue of an amino acid or amino acid d-erivative that preserves the immunoregulatory activity of said peptide; and R is a C1-C aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or C3-C cycloalkyl; in Ibm: X and Y are each Lys, Arg, His, or Orn; A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp, D-Ala, D-Val, D-Leu, D-Asn, D-Glu; D-Ser, D-Thr, or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a sjbstituent of the form R or RCO; B is Val, Leu, Glu, Gln, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Thr, Phe, lys, Orn, Trp, or a residue of a D-enantiomer of one of the foregoing amino acids; L, WO 86/04334 PCT/EP86/00012 44 C is OH, NH 2 NHR, NR 2 OR, Gly, Ser, Ala, VIal, Leu, D-Gly, D-Ser, D-Ala, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2

NHR,

NR 2 or OR; and R is a C I- C 8 aliphatic, C 6

C,

4 aryl, aralkyl or alkaryl, or C 3

C

1 4 cycloalkyl; in Ibn: X and Y are each Lys, Arg, His, or Orn; A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp or a residue of a derivativie of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO; B is VIal, Leu, Glu, Gin, Ser,. Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala; C is OH, NH 2 NUR, Gly or Ser; and R is a C 1-C alIk yl1; in Ibo: X is Lys, Arg, His, or Orn; Y is Lys, Arg, or His; A is Ac, Cly, Ala, Val, Asp, Asn, Glu, Arg, Ser, Thr, D-Asp, Ac-Asn, Val-Asp-, Gly-Asp-, Ala-Asp-, Ser-Asp-, Thr-Val-Asp-, or Leu-Thr-Val-Asp-; B is Val, Ser, Thr, Gly, Ala, Asp, Lys, or D-Ser; and C is OH, or NHR, where R is H or C 1

C

8 alkyl; I WO'86/04334 PCT/EP86/00012 in Ibp: X and Y are each Lys; A is H; 8 is Val, Leu, Glu, Gin, Ser, Thr. Gly, Ala, Pro, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, D-Aia, or D -Ser; and C is OH.

in Ibq: X and Y are each Lys; 'A is H; B is Val, Leu, Giu, Gin, Ser, Giy, Pro, Tyr, Phe, Lys, Trp, or D-Vai; and C is NHR, where R is H or a C I- C 8 aikyl; in Ibr: X

Y

A

B

C

Lys Arg H Ser, Thin, Gly, or Ala; and OH or NHR, where R is hydrogen or a CI a.1k yl in Ibs: X

Y

A

B

C

Arg; L ys; VIal, Ser, Thr, Gly, or Ala; and OH or NHE~, where R is H or a C 1 -C B alkyl; in Ibt: X is Ar Y is Ly A is H; B is As C i~ O0H g;

S;

p or Ser; or NHR, where R is H or a C*1-C 6 alkyl; -7 in Ibu: X

Y

A

B

C

PCT/EP86/OO 12 I I L ys Lys or Arg; Gly, Ala, Ser, Thr, k/al, or Ac-Asn; k/al; and 01H or NHR, where R is H or a C 1- C8alkyl; Lys or Arg; Ala, V/al, Ser, Thr, or Ai.-Asn; Thr and OH or NHR where R is H *or a C lC 8 alIk yl; in Ibv: X y

A

B

C

in Ibw: X is Lys Y is Lys or Arg; A is Ac-, Ala, Asn, Glu, Gly,,A-rg, Ac-Asn, Gly-Asp-, Ala-Asp-, kal-Asp-, Ser-Asp-, or Thr-k/al-Asp-; B is Ser; and C is OH or NHR, where R is H or a I.C I- 8akl in Ibx: X and Y( are residues of. amino acids or amino acid derivatives with positively charged side chains; A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N43-substitution of an amino acid with a substituent of the form R or RCO, provided that A is not Thr if X is Lys and, Y is Arg or a derivative of Arg; B is k/al, Leu, GIan, Set, Th1-r, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys, Sar, D-Thr, or D-Pro; C is OH, NH 2 NHRI NR 2 1 OR, Lily, Ser, Ala, k/al, Leu, D-Ser, D-Ala, D-k/al, D-Leu, or a residue of an

I

I O 86/04334 PCT/EP86/00012 47 amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C 1

-C

8 aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or a C -C14 cycloalkyl; in Iby: X and Y are residues of amino acids or amino acid derivatives with positively charged side chains: A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by Ni-substitution of an amino acid with a substituent of the form R or RCO; B is Glu, Asn, Orn, Trp, D-Val, D-Ser, D-Ala, D-Leu, D-Glu, D-Gln, D-Ala, D-Asn, D-Asp, D-Arg, D-His, D-Ile,- D-Tyr, D-Phe, D-Lys, D-.Orn, D-Trp,.or a residue of a derivative of one of the foregoing amino acids; C is OH, NH 2 NHR, NR 2 OR,Gly, Ser, Ala, Val, .Leu, D-Ser, D-Ala,'D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C1-C8 aliphatic, C 6

-C

1 4 aryl, aralkyl or alkaryl, or a C 3-C14 cycloalkyl; in Ibz: X and Y are residues of amino acids or amino acid derivatives with positively charged side chains; A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by Nd-substitution with a substituent of the form R or RCO; WO 86/04334 PCT/EP86/00012 48 B is V/al, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys, Urn, Trp, D-\Ial, U-Ser, or U-Ala; C is OH, NH 2 NHR, Gly or Ser; and R is a C C alkyl; in I bza: X and Y are each Lys, Arg, His, 0 n, D-Lys, D-Arg, U-His or U-Urn; A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N substitution of an amino acid w'ith a substituent of the form R or RCO, provided that A is not Thr if X is Lys and Y is Arg ar a derivative of Arg; B is k/al, Leu, Gin, Sqr, Thr, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys,.Sar, U-Thr, or U-Pro; C is NH 2 NHR, NR 2 OR, Gly, Ser, Ala, V/al, Leu, U-Ser, U-Ala, U-k/al, U-Leu, or a residue of an amino acid am'ide or ester formed by substitution of one of the foregoing amino acids with a substituent of the forni NH 2 NHR, 'R2' or OR; and R is a C 1

C

8 aliphatic, C 6

""C

1 4 aryl, aralkyl or alkaryl, or a C 3

C

1 4 cycloalikyl; in Ibzb.X and Y are each Lys, Arg, His, Urn, D-Ly-,, U-Arg, U-His or U-Urn; A is H, R, RCO, a residue of an -amino acid or amino acid derivative formed by N -substitution of anI amino acid with a substituent of the form, R or RCO; WO086/0' ii 20 $334 1k Th PCT/EP86/00012 B is Glu, Asn, Orn, Trp, 0-Val, D-.Ser, 0-Ala, D-LeU, D-Glu, 0-Gin, 0-Ala, D-Asn, 0-Asp, 0-Arg, 0-His, DvIE D-Tyr, 0-Phe, D-Lys, rn, D-Trp, or a residue of a derivative of one of the foregoing amino acids; Sis OH, NH 2 NHR, NR, OR, Gly, Ser, Ala, VJal, Leu, D-Ser, 0-Ala, 0-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino aci4 s with a substituent of the form NH 2 NHR, NR 2 or OR; and R is a C 1- C 8 aliphatic,. C 6- C4 aryl, aralkyl or alkaryl, or a C 3 C 14 cycloalkyl; in Ibzc: X and Y are eacm Lys, Arg, His, Orn, 0-Lys, D-Ard, D-His, or D-Orn; A is H, R, RCO, Asp, Gly, Ala, VIal, Leu, Asn, Glu, Ser, 0-Asp or a residue of a derivative of- one of the foregoing amino acids formed'by N -substitution with a substituent of the form R or RCO; B in~ Val, Leu, Glu, fln, Ser, Thr, Gly, Ala, Asn, kp Arg His Ilie, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala~l C is OH, NH 2 NHR, Gly or Ser; and R is a C 1

C

8 alkyl in Ibzd X and Y are each Lys, Arg His or Orr A is H, R, RCO, a residue of an amino acid or amino acid der'ivative formed by W'~substitution of an amino acid with a substituent of the form R~ or RCO, provided that A is not Thr if X is Lye and Y is Arg or a derivative of Arg; 1111 J1111 11111 1.8_ iiiiLjI!lI±±H.4 ZXMAnsNdNW1NH!DS(O:)v id Ot 1.

WO 86/04334 PCT/EP86/00012 B is V/al, Leu, Gin, Ser, Gly, Ala-, Asp, Arg, His, Ile, Tyr, Phe, Lys, Sar, D-Thr, or 0-Pro; C is OH, NH 2 NHR, NR 2 OR, Gly, Ser, 'Ala, Val, Leu, D-Ser, D-Ala, 0-k/al, D-Leu, or a residue of an amino acid amnide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 NR 2 or OR; and R is a C 1-C aiiphatic, C 6

C

1 4 aryi, araikyl or aikaryi, or a C 3

C

1 4 cycioaikyi; I i0 in Ibze: X and Y a3re each Lys, Arg, His, or Orn; A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N -substituti'on of an amino acid with a gubstituent of the form R or RCO; B is Giu, Asn, Orn, Trp, D-k/ai, D-Ser, 0D-Aia, 125 D-Leu, D-Gilu, 0-Gin, D-Aia, D-Asn, 0-Asp, D-Arg, 0-His, D-le, D-Tyi D-Phe, 0-Lys, D-Orri, D-Trp, or a residue of a derivative of one of the foregoing amino acids; C is OH, NH 2 NHR, NR 2 OR, Gly, Ser, Aia, V/ai, Leu, D-Ser, 0-Ala, 0-k/ai, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent the form NH 2 NHR, NR 2 1 or OR; and R is a C 1

C

8 aliphatic, C 6

-C

1 aryi, araikyl or aikaryl, or a C 3

C

1 4 cycloalkyl;p 11 V6 86/04334 ~v 86/4334PCT/EP86/000 12 51 in Ibzf: X and Y are each Lys, Arg, [is, or Orn; A is H, R, Rf2O, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, D-Asp or a residue of a derivativje of one of the foregoing amino acids formed by N -substitution with a substituent of the form R~ or RCO; B is VJal, Leu, Glu, Gin, Fier, Thr, Gly, Ala, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Lirn, Tr'p, D-Val, D-Ser, or D-Ala; C is OH, NH 2 NHR, Cly or Ser; and R ii a C 2 C 8 aikyl; in Ibzg: X is Lys, Arg, His, or Orn; Y is Lys, Arg, or His; A is Ac, Giy, Ala, VIal, Asp, Asn, Giu, Arg, Ser, Thr, D-Asp, Ac-Asn, Val-Asp-, Giy-Asp-, Ala-Asp-, Ser-Asp-, i~Thr-Val-Asp-, or Leu-Thi'-Val-Asp-; B is VJal, Ser, Thr, Gly, Ala, Asp, Lys, or D-Ser; and C is OH, or NHR, where R is H or C 1

C

3 alkyl; in Ibzh: X aid Y are each Lys; A is H; B is Val, Leu, Glu, Gin, Ser, Thr, Ala, Asn, Arp, Arg, His, Ile, Tyr, Phe, Lyka, Orn, D-Aia, or D-Ser; and C is OH; in Ibzi: X and Y are each Lys; A is H; WO 86/04334 PTE8/01 PCT/EP86/00012

B

Lys, Trp,

C

in Ibzj :X

Y

A

B

C

is VSal, Leu, Giu, Gin, Ser, Gly, Pro, Tyr, Phe, or D-Vai; and is NHR, where R is H or a C 1 c 3 ai1k yl1 is Lys; is Arg; is H; is Ser, Thr, Gly, or Ala; and is OH or NHR, where R is H or a C 1-c alkyl; in Ibzk: X is Arg; Y is L y s A is RH; B i s Vali Ser, Thr, Giy or Ala; and C is OH or NHR, where R is hydrogen or a lower aikyl; in Ibzl:X

Y

alkyli; is A r g is Ivs; A 1: H is Aksp or Ser; is OH or NHR, where R is hydrogen or a C 1- C 3 is Lys; is Lys or Arg; is Gly, Ala, Ser, Val, or Ac-Asn; is VSal; and is OH or NHR, where R is hydrogen or aC -3 in Ibzm:X

Y

a Ik y 1; Sd 86/04334 PCT/EP86/00012 in Ibzn: in Ibzo: X is Lys; Y is Lys or Arg; A is Ala, Val, Ser, or Ac-Asn; B is Thr; and C is OH or NHR, where R is H or a C 1

-C

3 alkyl; X is Lys; Y is Lys or Arg; A is Ac, Ala, Asn, Glu, Gly, Arg, Ac-Asn, Ala-Asp-, Val-Asp-, Ser-Asp-, or Thr-Val-Asp-; B is Ser; and C is OH or NHR, where R is H or a C 2

-C

3 alkyl.

Gly-Asp-, The peptides of formula I can be prepared by usual methods of peptide synthesis.

In particular, a peptide of formula I is prepared by liberating it from its corresponding functional derivative which may be bound by .a covalent bond to a solid resin by treatment under acidic or basic conditions, and, if desired, the peptide thus obtained is esterified, amidated and/or acylated to yield a corresponding ester, N- and/or 0-acyl derivative or is transformed into one of its pharmacologically acceptable salts by treatment with a base or an acid.

WO86/04334 PCT/EP86/00012 WO 86/04334 54 Immunoregulatory Activity of the Subject Peptides As discussed above, T-lymphocytes (T cells) play a powerful role in the in vivo induction, suppression, and regulation of immune responses leading to a number of autoimmune and other disease conditions. In order to examine the effect of peptides or other drugs on T cell function, in vitro assays have been developed which are predictive of in vivo I function.

In the usual in vitro assays of T cell function, .ymphoid cells from peripheral b ood or from the spleen, lymph nodes or other organs are isolated and cultured with or without potential immunoregulatory druls. The,extent to which lymphocyte function in general and T cell function in particular are regulated may be assessed by many measurement techniques. The most common technique measures the amount of a radioactive precursor of DNA (tritiated thymidine) incorporated into newly synthesized DNA of lymphocytes. Since only "activated" cells that are preparing to divide synthesize new DNA, the amount of tritiated thymidine incorporated in cells and the resultant incorporated radioactivity is directly related to the extent of cell activation and resultant proliferation.

I

1 croaeinclsadterslaticroaeraiatvy I' W 86/04334 PCT/EP86/00012 In order to predict whether a potential immunoregulatory drug will be useful in treating a particular disease or class of diseases, it is important that it be tested in in vitro or in vivo system that are reflective of the particular disease or diseases in question. Several in vitro assays of T cell function in particular are highly predictive of in vivo efficacy in treating organ transplantation rejection and autoimmune disease.

The mixed lymphocyte response (MLR) is an in vitro analog of the in vivo organ transplantation rejection response.

To perform the MLR, T cell-containing lymphocyte preparations from two humans or animals are cultured together for five to seven days under standard cell culture conditions. During the Sculture, T cells from both subjects re'cognize histocompatibility molecules of the other subject. If both subjects are genetically identical, their histocompatiblity molecules will also be identical and no activation or cell proliferation will occur. The radioactivity incorporated into cells will therefore be very low since few cells are stimulated to divide. By contrast, if both subjects are genetically non-identical, both subjects' cells will recognize the foreign histocompatibility molecules of the other and will divide and proliferate. The level of cellular radioactivity will therefore be increased over that of control, unstimulated cultures and will be directly proportional to the magnitude of the genetic difference between the two subjects. This assay is termed a two-way MLR since each subject's cells respond to the other's proliferative stimuli. If one of the subject's cells is prevented from dividing by, for example, exposure to gamma ir- WO 86/04334 PCT/EP86/00012 56 radiation or mitomycin C, then the observed proliferation and incorporated radioactivity will be due only to the other subject's cellular proliferation. This assay is termed a one-way

MLR.

During a MLR, some of the proliferating T cells differentiate into T cells aule to kill the target cells which produced the initial stimulus. These sensitized cytotoxic or Killer T cells are primarily directed toward the target cell's histocompatibility molecules and produce T cell-mediated destruction which closely resembles the destruction which occurs during organ transplantation rejection. If T cells from a transplant recipient are sensitized to the histocompatibility antigens of a transplanted organ in vitro and then injected into the recipient, the sensitized T cells will cause the organ to be rejected. Sensitized T cells alone are therefore both-necessary and sufficient to cause organ transplant rejection. As demonstrated in Table 1, the peptides of the present invention can substantially suppress the MLR without significantly affecting cell viability. This indicates that such peptides have therapeutic usefulness since they can suppress the immune response responsible for the immunologic rejection of transplanted organs and autoimmune diseases.

It should be noted that suppression of T cell activities in vitro by peptides of the present invention is but one manifestation of the complex immnoregulation which occurs in vitro and in vivo and which leads to reduction or prevention of autoimmune disease or of the rejection of transplanted organs. For example, while the peptides of the present invention can suppress a MLR at

-C--I

SW6 86/04334 PCT/EP86/00012 57 concentrations listed in table 1, lower peptide concentrations typically result in stimulation of tritiated thymidine uptake which is reflective of the peptides' ability to activate certain immunoregulatory processes.

Administration of the subject peptides to animals, for example, can in certain cases result in stimulation of certain T lymphocyte subsets, which can in turn lead to net suppression of a clinical disease or condition. It is thus entirely within the scope of the peptides of the present invention that certain immunostimulatory properties, in addition to the suppressive properties described herein, will be expressed in alternate in vitro and in vivo tests of the subject peptides.

The MLR is a specific example of a more general T cell response toward "foreign" antigens. In a MLR, T cells recognize and proliferate in response primarily to antigens located on histocompatibility molecules of the stimulator cells. Molecules unrelated to histocompatibility antigens may also serve as potent stimulators of T cell proliferative and killing responses. In fact virtually any molecule, if presented to T cells in an appropriate manner, may stimulate T cell proliferative, regulatory and killing responses directed toward the stimulating antigen. Such antigen-specific T cell responses in humans are thought to be responsible for many of the autoimmune diseases listed earlier when immune tolerance is reduced or lost.

Specific in vitro assays exist which can measure a T cell response directed toward any specific antigen. In WO 86/04334 PCT/EP86/00012 58 particular, the therapeutic and immunoregulatory utility of the peptides of the present invention is further demonstrated by an antigen-specific T cell proliferation assay which uses mice that have been immunized with a particular antigen such as Bovine Serum Albumin (BSA). Upon immunization, T cells in lymph nodes wnich drain the injection site are activated and stimulated to proliferate. During this immune response, regulatory and Killer T cells which specifically recognize BSA are generated. At six days post-immunization when the T cell response is sufficiently mature, the draining lymph nodes are removed and cells eluted from them are placed into a standard culture. BSA or a second, unrelated antigen is then added to the cultures with or without the immunoregulatory peptides of the present invention. Quantitation of antigen-induced T cell activation and proliferation is assessed by cellular tritiated thymidine incorporation at the end of the culture incubation in a manner similar to that used in MLR cultures. Cultures containing BSA will produce high levels of tritiated thymidine incorporation because the BSA-sensitized T cells recognize the BSA molecules and become stimulated to proliferate. Cultures containing an unrelated antigen, by contrast, have low stimulation levels because they lack receptors directed toward the unrelated antigen.

As demonstrated in Table 2, the peptides of the present invention substantially suppress the T cill proliferative response toward specific molecular or cellular antigens without significantly affecting cellular viability. This observation is 4_^ W086/04334 PCT/EP86/00012 59 of therapeutic importance because it indicates that such peptides can suppress an abnormal T call immune response directed toward a wide range of unrelated antigens different from those found on histocompatibility antigens. As previously discussed, abnormal Killer T cell responses directed against either foreign or normal "self" antigens on various organs are thought to be responsible for many autoimmune diseases. Other autoimmune diseases may result from an abnormal regulatory T cell response towards specific antigens that resulb in an imbalanced response of other T cell-regulated portions of the immune system.

It is possible to develop in vivo models of human autoimmune disease in which animals are immunized with organ tissues from healthy genetically identical animals. Animals immunized against one of their organs develop an autoimmune response which may result in destruction of the organ. T cells isolated from such an immunized animal may be very specific for molecules and cells of the injected organ showing that the T cells have receptors for organ-specific antigens.

By using organs or their preparations as immunizing agents as described, it is possible to develop animal models of autoimmune diseases involving virtually any organ system, some of which were previously enumerated.

The peptides of the present invention are efficacious in reducing or preventing the tissue destruction characteristic of autoimmune disease in vivo. Figures 1 and 2 demonstrate the efficacy of one peptide and one of its peptidomimetic analogs in WO 86/3 PCT/EP86/00012 WO 86/04334 treating an animal model of human multiple sclerosis In this model, termed Experimental Allergic Encephalomyelitis (EAE),.

SJL/J mice were immunized with a homogenate of spinal cord from SJL/J mice in a Complete Freund's Adjuvant medium. This homogenate contains cells and molecules representative of the many potential antigens which are present in the central nervous system (CNS). Such animals typically develop vestibular and I occular disturbances, weakness, paralysis, and other signs of central nervous system destruction characteristic of both EAE and MS ten to fifteen days after initial immunization. The EAE model is discussed in Brown et al., Lab. Invest. 45(3):278-284 (1981).

S Like the majority of human MS cases, the disease severity in this animal MS model waxes and wanes during the disease course. Mice receiving onlysaline injections two to three times per week exhibit high peaks and troughs of disease severity. By contrast, mice receiving peptide or peptidomimetic demonstrated very mild disease initially and were clinically disease fr-e by the study's end. Microscopic analysis of brain sections from both peptide and saline-treated mice is presented in Table 3.

One hundred percent of saline-treated mjce demonstrated substanf tial numbers of visible lesions which represent physical brain destruction in all portions of the CNS examined. By contrast, sixty percent of peptide-treated mice were lesion-free while the remaining forty percent of peptide-treated mice had few, scattered lesions confirming the clinically-elicited observations.

4A WO 86/04334 PCT/EP86/00012 61 In addition to the immunoregulatory properties of the peptides of the present invention, the peptides may also be used to regulate the growth of neoplastic (cancer) cells in vivo. All cancer cells have in common the property of unregulated cell replication. Whereas normal cells replicate at a rate consistent with the normal functioning of the organ in which the cells reside, cancerous cells replicate in an unregulated manner, which leads to tumor formation and possible destruction of adjacent healthy tissue.

As demonstrated in Tables 4 and 5, the peptides of the present invention can suppress or prevent the growth of a variety of neoplastic cells in vitro. Figure 3 demonstrates the anticancer properties of one of the peptides of the present invention in mice with lymphoma/leukemia of T cell origin. Whereas saline treated mice had a median survival of 23.5 days after injection of lymphoma/leukemia cells, peptide-treated mice had a median I survival of 30.5 days. Additionally, all saline-treated mice I were dead by day 31 while one peptide-treated mouse was tumor-free at day The peptides of the present invention may be administered to humans or other mammals by a variety of means commonly utilized with respect to other therapeutic agents. Injected subcutaneously, a dosage range of from about 25ug/kg to about mg/kg of body weight may be employed, with a preferred range of about 2.5 to 50 mg/kg. Sterile saline, aqueous dextrose and 'tw-f: ICLI---- e~ i- WO 86/04334 PCT/EP86/00012 62 glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions. Intravenous, intraperitoneal, and intramuscular injections may also be used at dosages comparable to those of subcutaneous injections. The peptides may be administered orally at dosages generally ranging from one to 100 times that of an injected dose, although greater dosages may be required depending on factors such as f&od intake. The peptides may be combined with appropriate pharmaceutical carriers in the form of pills, capsules, elixers, suspensions, aerosols, powers, or in other forms. Intranasal administration may be achieved by using an appropriate amphipathic vehicle such as, for example, glycocholic acid or EDTA. Administration may be achieved topically or transdermally, using an appropriate carrier in the form of for example, an ointment. Sublingual, rectal, or topical ocular administration using appropriate pharmaceutical media may be employed. Sustained-release formulations using, for example, liposome delivery media, are also appropriate vehicles for administering the subject peptides.

r/

Z~

Wd 86/64334 PCT/EP86/OO 12 63 TABLE 1 PERCENT INHIBITION OF DNA PROLIFERATION OF MURINE AND HUMAN LYMPHOCYTES DURING MIXED LYMPHOCYTE CULTURES IN THE PRESENCE OF THE SUBJECT PEPTIDES Murine MLR Human MLR i eptidel Peptjde Peptide Sequence Inhib!/ (ug/mi) InZib!/ Lys-NH2 100 98 200 99 D-Lys-NH 2 50 99 200 99 D-Lys-NH 200 99 400 99 DL0 HCH CH 3 0 9408 Lys-Ser 50 99 200 99 kD-Lys-Ser 5 0 99 400 14 Ly5-'Ser-NH 2 s0 99 100 99 desamino-Lys-Ser 200 98 400 99 Ac-Lys-Ser 50 '99 400 71 Ac-Lys-Ser-NH 2 400 92 400 44 200 96 400 97 Orn-Ala, 50 99 100 91 Orn-D-Ala 50 98 100 94 1rn-Gl0 10 99' 200 97 Atg-D-Ala 200 j9 400 88 Arg-Sar 200 98 400 97 Arg-Thr 400 83 50 -32 His-Ser 100 99 400 97 Lys-Cys 200 99 100 93 Lys-Phe 100 99 400 99 Lys-D-Phe 400 -6,1 400 54 Lys-Tyr 200 99 400 Lys-Asp 100 99 100 -41 Lys-Gli 100 99 400 Lys-Leu 100 99 200 26 Lys-Pro 200 99 200 -19 Lys-HomoSer 200 99 200 93 Aia-Lvs-Ser 50 99 50 Alai-Lys-Gly 50 99 200 D-rla-D-Lys-D-Ser 50 96 200 Gly-Lys-Ser 200 89 400 94 Val -Lys-Ser 100 -59 400 99 Ph..-Lys-D-Ala 50 99 E PhE-Lys-Ta 400 99 100 -100 Tyr-Lyts-Gly 200 99- 400 94 Asp-Lys-Ser 50 09 400 99 (Table 1 continues next page] 1; WO 86/04334 PCT/EP86/000 12 64 (Table 1, continued] Murine MLR Human MLR (peptide7 (Pept d67 Peptide Sequence (ug/ml t Inhi./ (uE/ml) tnhibl/ Sar-D-Lys-Ser 100 99 400 58 Lys-Ser-Tyr 200 95 400 99 Lys-Ser-Ala 200 98 400 99 Phe-Lys-Ser-Tyr 200 98 400 96 Lys-Val-Lys 400 99 100 96 Lys-Glu-Lys so 99 400 84 Lys-iln-Lys 100 98 100 Lys-Arg-Lys 100 99 100 76 Ays-His-Lys 100 99 100 77 Lys-D-Ala-Lys 200 99 200 43 Lys-Val-Lys-N 7 100 99 1'0 96 Lys-Leu-Lys-NF.

2 50 99 100 89 Lys-Pro-Lys-NH 2 50 99 100 92 Lys-Tyr-Lys-NH 2 100 99 400 098 Lys-D-Val-Lys-NH 2 400 99 400 63 Lys-Ser-Arg 100 92 100 49 Lys-Thr-Arg 200 98 200 -47 Arg-Gly-Lys 200 98 400 -31 Arg-Ala-Lys 100 92 50 -39 Arg-Asp-Arg 50 99 200 92 Ac-Lys-Ser-Lys 400 97 400 99 Val-Lys-Val-Lys-NH 2 100 99 200 99 Ala-Lys-Val-Lys-NH 2 50 98 100 Ala-Lys-Ser-Arg 400 99 400 -132 Arg-Lys-Ser-Arg 100 96 100 -141 Asn-Lys-Ser-Arg 50 70 400 99 Ac-Asn--Lys-Ser-Arg 200 99 400 99 Gly-Asp-Lys-Ser-Arg 100 90 400 51 Asp-Lys-Lys-Arg 100 99 100 86 Asp-Lys-Ile-Arg 200 99 400 42 Ser-Lys-Val-Arg 100 98 400 Gly-Lys-Val-Arg 100 99 100 Asp-Orn-Ser-Arg 100 99 200 99 Asp-His-Ser-Arg 400 95 400 -36 Asp-Lys-Ser-His 100 90 400 26 /Table 1 continues next pagu, CU~*i\ ,i i 'WO 86/04334 PCT/EP86/00012

IA

'i

I

iA /Table 1, continued/ 1/ Percentage inhibition as compared to cultures in the absence of peptide.

NOTE: Splenocytes from Balb/c mice were cocultured with splenocytes from C57B/6 mice and one healthy human donor were cultured with irradiated stimulator cells from a second healthy human donor for seven days at 370C and 5% CO 2 Cultures were pulsed with 1 uCi R-thymidine for six hours.

The concentrations of the subject peptides which produce inhibition or stimulation of proliferation may occasionally vary from the concentrations listed depending on many factors including age of the donor, time of day of blood drawing, food' intake and other factors.

WO 86/04334PC/P001 66 TA&SLE 2 INHIBITION OF ANTIGEN-SPECIFIC DNA PROLIFERATION OF SENSITIZED MURINE LYMPHOCYTES IN THE PRESENCE OF THE SUBJECT PEPTIDES Concentration Peptide Sequence (lig /ml) I I~hibition.- Lys-NH 2 100 98 D-Lys-NH 2 100 99 D-Lys-NH 2

CH

2

CII

3 100 36 Lys-Ser 50 99 -Lys-Ser 12596 Lys -Se r-NN 2 25 92 I des amino -tyo -Se r 100 68 Ac-Lys-Ser 5098 Ac-Lys-Ser-NH 2 50 99 N-Ch 3 -Lys-D-Ser 100 99 Orn-D-Ala 50 99 Orn-Gly 12.5 94 Arg-D-Ala 12.5 36 Arg-Sar 100 99 Arg-Thr )5 22 His-Ser 100 -13 Lys -Cys 100 74 Lys-Phe 50 99 F 100 25Lys-Asp 100 92 Lys-Pro 10 0 44 Ala-Lys-Ser 5o 98 Ala-Lys-Gly 100 99 D-Ala-D-Lys-D-Ser 100 -31 N 30 Phe-Lys-D-Ala 100 99 Asp-Lys-Ser 100 99 Sar-D-Lys-Ser 100 18 Lys-Ser-Tyr 100 Lys-Ser-Ala 12.5 -57 /Table 2 continues next pagje7 -7 NWv 86/04334 PCT/EP86/000 12 /Table 2 continuedj Concentration (W.g/m1) Inhibition- Peptide Sequence Lys-Val-Lys Lys-Glu-Lys Lys-Arg-Lys Lys-His-Lys Lys-D-Ala-Lys Lys-Va:L-Lys-NH 2 Ly s-L e U-Ly5-

NH

2 Lys-Thr-Arg Atg-Gly-Lys Arg-Ala-Lys Arg-Asp-Arg Val-Lys-Val-Lys-NE 2 Ala-Lys-Val-Lys-NH 2 Asp~-Lys-Ser-Arg Asp-Orri-Ser-Ar'g 50 12.5 50 100 100 50 50 25 -96 98 100 99 62 97 94 -7 7 99 99 99 99 .94 100 25 50 50 100 25 l/

NOTE:

Percentage inhibition. as compared to cultures in the absence of peptide.

Balb/c mice were immunized with ovalbumin (OVA) in Complete Freund's Adjuvant. Seven days later lymphocytes from superficial inguinal lymph nodes were cultured with 100 ug/ml OVA and peptide. Cultures were pulsed with 1 UCI 3 H-thymidine for 24 hours from day 4 to day The concentrations of the subject peptides which produce inhibition or stimulatio~n of proliferation may occasionally vary from the concentrations listed depending on many factors including age of the donor, time of day of blood drawing, food intake and other factors.

i i ~r'-Y'IO LnfLfhI I WO 86/04334 re i/Lroo/uuu 68 TABLE 3 PATHOLOGICAL LESIONS PRESENT IN CENTRAL NERVOUS TISSUE OF MICE DURING EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS TREATED WITH SALINE OR SUBJECT PEPTIDE LYS-SER 1.

SUBCUTANEOUS INJECTIONS* CNS TISSUE Spinal Cord Cerebellum Brainstem SALINE (8) LYS-SER 8.1 3.9 20.1 6.7 12.3 6.5 2.6 2.1 2.8 0.9 4.6 2.2 1.2 1.0 0.6 0.4 (97) 0.4 0.2 (97) 1.2 0.9 (54) 1.8 1.2 (36) 1.3 0.9 (72) Basal Nuclei Cerebral White Matter Cerebral Grey Matter Mean standard error of lesions detected per mouse.

NOTE: SJL/J mice were injected subcutaneously with mouse spinal cord homogenate in Complete Freund's Adjuvant on days 0 and 7 and injected with saline or 1 mg peptide 3 times per week from day 11 to 32. The number of mice in each group is enclosed within parentheses. Tissue was sectiotned, the sample identity was coded and was examined microscopically by an uninformed observer. The percent inhibition of lesions is given in the last column in parentheses.

p'r/Ep86/0012 Wd 86/04334 69 TABLE 4 PERCENT INHIBITION OF DNA PRQLIFERATYON OF MURINE LEUKEMIC CELLS IN THE PRESENCE OF THE SUBJECT PEPTIDES L 1210 Cells WEHI-22 Cells Peptidel [Peptide) Peptide Sequence LiZ.2 Inhib.!/ (ug/ml) Inhib I 100 98 200 24 D-Lys-NH 200 98 200 72 Lys-Ser 200 78 200 37 D-Lys-Ser 200 -13 200 26 desamino-Lys-Ser 200 21 200 3 Ac-Lys-Ser 200 44 200 Ac-Lys-Ser-NH 50 -57 200 18

N-CH

3 -Lys-D-Ser 200 37 200 74 Orn-Ala 100 71 100 81 Orn-D-Ala 100 89 100 86 Orn-Gly 192 95 100 86 Arg-D-Ala 20u 89 200 ArA-Sar 200 97 100 77.

His-Ser 200 67 200 71 Lys-Cys 50 94 100 99 Lys-Phe 200 99 Lys-D-Phe 200 19 200 -27 Lys-Asp 200 38 200 8 Lys-Gin 200 -34 200 7 Lys-Leu 200 69 200 Lys-Pro i00 8 100 24 Ala-Lys-Ser 100 49 200 Ala-Lys-;cly 200 83 200 54 D-Ala-D-Lys-D-Ser 100 29 100 G1y-Lys-Ser 200 91 200 91 Val-Lys-Ser 200 80 200 Phe-Lys-Sar 200 16 200 17 Tyr-Lys-Gly 50 -18 100 -8 Asp-Lys-Ser 100. 39 Sar-'D-Lys-Ser 200 81 200 72 Lys-Ser-Tyr 100 Lys-Ser-Ala 200 98 200 89 Phe-Lys-Ser-Tyr 50 -7 50 /Table 4 continues next page! ~WO 86/04334PT/86OO1 3. 1 /Table 4, cont inuedl L 12.20 Cells [PeptdV 1 WEHI-22 Cells ug[Peptide]- _u/ml) Inhib../ Peptide Sequence Lys -Val1-Lys Lys-Glu-Lys Lys-Gln-Lys Lys-Arg-Lys Lys-His-Lys Lys-D-Ala-Lys Lys-Val-Lys-NH 2 Lys-Leu-Lys-NH 2 Lys-Pro-Lys-NH 2 Lys-Tyr-Lys-NH, Lys-D-Val-L ysIFH 2 Lys-Ser-Arg Lys-Thr-Arg Arg-Gly-Lys Arg-Ala-Lys Arg-Asp-Arg Ac-Lys-Ser-Lys Val-Lys-Val-Lys-NH-, Ala-Lys-Val-Lys-mH' Ala-ys-Sr-Ar Arg-Lys-Ser-Arg Asn-Lys-Ser-Arg Ac-As n-Lys-Ser-Arg Gly-Asp-Lys-Ser-Arg Asp-Lys-Lys-Arg Asp-Lys-Ile-Arg Ser-Lys-Val-Arg Gly-Lys-Val-Arg AsP-Orn-Ser-Arg Asp-His-Ser-Arg 100 200 200 200 200 25 200 200 100 200 200 100 200 200 100 100 200 200 64 43 99 73 99 -19 93 96 95 85 28 28 17 19 -13 88 65 200 200 200 100 200 100 100 200 100 200 100 100 200 200 100 200 200 200 100 100 100 100 100 100 100 100 100 200 100 43 94 94 -83 82 96 81 24 19 -22 94 58 76 16 -31 16 -41 -14 81 21 72 6 84 -14 200 50 98 -28 1/ Percentage inhibition peptide.

as compared to cultures in the absence of hours and then NOTE: Cells were cultursd at 379C and 5% CO 2 for 48 pulsed with 1 uCi H-thymidine for 6 hours.

The concentrations of the subject peptides which produce inhibition may occasionally vary from the concentrations listed depending on many factors including age of the cells, culture conditions and other factors.

8' e i,, I 6 86/04334 PCT/EP86/00012 TABLE PERCENT INHIBITION OF DNA PROLIFERATION OF HUMAN MENINGIOMA TUMOR CELLS IN THE PRESENCE OF THE SUBJECT PEPTIDES EFc Tumor Cells [Peptide] (ug/ml) Inhib.- LG Tumor Cells [Peptide] (ug /ml) Inhib.

Peptide Sequence Lys-NH 2 D-Lys-NH 2 Lys-Ser Lys-Ser-NH 2 desamino-Lys-Ser Ac-Lys-Ser-NH 2

N-CH

3 -Lys-D-Ser Orn-D-Ala Orn-Gly 200 200 200 100 200 200 100 200 200 Arg-D-Ala Arg-Sar His-Ser Lys-Cys Lys-Phe Lys-Tyr Ala-Lys-Ser Ala-Lys-Gly D-Ala-D-Lys-D-Ser Gly-Lys-Ser Phe-Lys-D-Ala Lys-Ser-Tyr Lys-Ser-Ala Lys-Arg-Lys 200 200 200 200 100 200 200 50 200 50 200 100 200 100 200 200 37 82 31 91 .63 46 90 79 23 -12 200 200 200 100 200 50 100 200 100 200 100 200 100 200 50 200 50 84 96 88 78 -439 52 48 97 S77 -42 72 53 100 200 200 200 Lys-Val-Lys-NH 2 Lys-Ley-Lys-NH 2 Asp-Lys-Lys-Arg 100 1/ Percentage inhibition peptide.

as compared to cultures in the absence of hours and then NOTE: Cells were cultured at 37*C and 5% CO 2 for 48 pulsed with 1 uCi H-thymidine for 6 hours.

The concentrations of the subject peptides which produce inhibition may occasionally vary from the concentrations listed depending on many factors including age of the -ells, culture conditions and other factors.

4, r WO 86/04334 PCT/EP86/o0012 72 EXAMPLE 1 Solution phase synthesis of Lys-Ser salts using t-butyloxycarbonyl protection. L-Serine (157.6 g, 1.5 moles) and sodium bicarbonate (168 g, 2 moles) were dissolved in distilled water (2 1) and treated with a solution of N,N'-bis-t-butyloxycarbonyl-L-lysine N-hydroxysuccinimide ester (444 g, 1 mole) in tetrahydrofuran (2 1).

The resulting solution (pH approx. 9) was stirred overnight at room temperature. The pH was then adjusted to 6.5 with 1 N sodium bisulfate and the mixture evaporated to approximately half the volume under reduced pressure. The residue was extracted twice with ether before adjusting the pH to 3.0 (1 N sodium bisulfate) and extracting the product three times with ethyl acetate (2 1, 1 1, 1 The combined organic extracts were washed with 5% brine and :hen dried over 'sodium sulfate. The solution was then concentrated under reduced pressure to approximately 1.5 1, dicyclohexylamine (199.2 ml, 1 mole) added and the mixture stirred at room temperature for 0.5 hours. The product, N,N'-bis-t-butyloxycarbonyl-L-lysyl-L-serine dicyclohexylammonium salt, crystallized out of the solution and was recrystallized from isopropanol. Yield: 464 g m.p. 126-129C, (a]2 5 +4.48° (c 1, ethanol).

The dicyclohexylammonium sat (73 g) was dissolved in icewater (800 ml), the pH adjusted to 3 withn i N sodium bisulfate and the product extracted into ethyl acetate (500 ml, 209 ml, 100 ml). The combined organic extracts were dried over sodium sulfate and the solution evaporated to give N,N'.-bis-t-butyloxycarbonyl-L-lysyl-L-serine (46.3 g).

WO 86/04334 PCT/EP86/00012 73 Deprotection to bis-trifluoroacetate salt. N,N'-bis-tbutyloxycarbonyl-L-lysyl-L-serine (17.8 g) was dissolved in dichloromethane (70 ml), anhydrous trifluoroacetic acid (50 ml) added and the mixture stirred at room temperature for 0.5 hours. The solution was evaporated under reduced pressure and the residue triturated three times with dichloromethane and three times with ether to give the bis-trifluoroacetate salt of L-lysyl-L-serine in quantitative yield as a colorless, hygroscopic solid.

Deprotection to dihydrochloride salt. N,N'-Bis-t-butyloxycarbonyl-L-lysyl-L-serine (20 g) was dissolved in an anhydrous solution of hydrogen chloride in ethyl acetate (3.5 N, 200 ml) and the mixture stirred at room temperature for 0.5 hours. The solution was evaporated under reduced pressure and the residue triturated four times with eth6r to give the dihydrochloride salt of L-lysyl-L-serine in quantitative yield as a white hygroscopic solid.

Alternately, N,N'-bis-t-butyloxycarbonyl-L-lysyl-L-serine g) was dissolved in dioxane (50 ml) and treated with a solution of hydrogen chloride in anhydrous dioxane (4 N, 250 ml). The mixture was stirred at room temperature for 0.5 hours, tvaporated under reduced pressure and the residue triturated four timas with ether to give the dihydrochloride salt of L-lysyl-L-serine in quantitative yield as a colorless, glassy hygroscopic solid.

4k WO 86/04334 PCT/EP86/00012 74 EXAMPLE 2 Solution phase synthesis of Lys-Ser salts using benzyloxycarbonyl protection. Following the general procedure described in Example 1, L-serine, (15.76 g, 0.15 mole) and sodium bicarbonate (25.2 g, 0.3 mole) were dissolved in distilled water (325 ml) and treated with a solution of N,N'-bis-benzyloxycarbonyl-L-lysine N-hydroxysuccinimide ester (51.56 g, 0.1 mole) in tetrahydrofuran (300 ml). After S stirring at room temperature overnight, the reaction mixture was concentrated under reduced pressure to approximately half the volume, extracted with ether (2 x 500 ml) and poured, with rapid stirring, into ice-cold hydrochloric acid (1 N, 3.5 The white precipitate which separated was quickly extracted into ethyl acetatt (3 1) and the organic phase washed with ice-cold 1 N HC1 (2 x 1 water (3 x 1 1) and dried over magnesium sulfate. The solution was evaporated to dryness under reduced pressure and the solid residue triturated with hexanes to give N,N'-bis-benzyloxycarbonyl-L-lysyl-L-serine as a colorless hygroscopic solid, m.p. 143-1450C. Yield: 45.03 g.

Conversion to dicyclohexylammonium salt. The product was converted to the dicyclohexylamine salt by dissolving in a minimum volume of ethyl acetate (approx. 7 1) containing methanol (1 1) and treating with i equivalent of redistilled dicyclohexylamine (17.9 ml). The crystalline product'was filtered, washed with ether, dried and recrystallized from isopropanol. The product was reconverted to the protected dipeptide by neutralizing with aqueous sodium bisulfate, as described in Example 1. Yield: 31.5 g.

'Vd 86/04334 PCT/EP86/ooo 12 Deprotection to acetate salt. N,N'-Bis-benzyloxycarbonyl-L-lysyl-L-serine (3 g) was dissolved in a mixture of methanol ml) and water (20 ml) containing acetic acid (0.35 ml, 2 equivalents) and hydrogenated over 10% palladium on carbon (1 g) at atmospheric pressure overnight. After removing the catalyst by filtration, the solution was cqncentrated to approximately half the volume under reduced pressure, and the residue lyophilized, to give the acetate salt of L-lysyl-L-serine in quantitative yield as a colorlcss, hygroscopic solid.

EXAMPLE 3 ted Automated solid-phase synthesis cf Lys-Ser on chloromethylatdpolystyrene resin. Tert-butyloxycarbonyl-amino acid resins, protected amino acid derivatives, and other peptide synthesis products may be obtained from, Peninsula Laboratories, Inc., 611 Taylor orBceInc., 3132 Kashiwa Street, TorneCA955 Ter v,-bu tyloxyca rbony 1-O-benzy 1-L-se r ine- res in (sustiutin a zv=oles/g; 4.0 g) wir~s deblocked and coupled with synthzz: zer (Beckman Model 990) programmed with the following protocol: 1. wash with dichloromethane (50 ml), 3 x 1 minute.

2. Deblock with trifluoroacetic acid (35% v/v in dichloromethane, S0 ml), 1 x 1 minute, I. x 20 minutes.

3. Wash with dichloromethane (50 ml), 3 x I minute; iorpnl(50 ml), 2 x 1 minute; and dichloromethane (50 ml), 3 x 1 WO 86/04334 PCT/EP86/00012 76 4. Neutralize with triethylamine (10% v/v in dichloromethane, 50 ml), 1 x 1 minute and 1 x 5 minutes.

Wash with dichloromethane (50 ml), 4 x 1 minute.

6. Couple with t-butyloxycarbonyl-e-benzyloxycarbonyl-L-lysine (2.59 g, 6.8 mmoles, 2 equivalents) in dichloromethafe (30 ml), together with N,N-dicyclohexylcarbodiimide (0.5 M in dichloromethane, 13.6 ml, 2 equivalents), 1 x 90 minutes., 7. Repeat step 3.

8. Repeat step 1.

9. Repeat step 2.

Repeat step 3.

(Note: Steps 1-7 constitute one complete coupling cycle.) The resin was collected on a sintered glass funnel, washed successively with ethanol and dichloromethane and dried under vacuum. Yield: 4.96 g.

The peptide was cleaved from the resin by treatment with liquid hydrogen fluoride (10 ml per gram of dry resin) in the presence of anisole (1 ml per gram of resin) and 0@C for one hour. After removal of the hydrogen fluoride under vacuum, ether (10 ml per gram of resin) was added, the resin transferred to a sintered glass funnel and washed alternately with ether (4 x 40 ml) and chloroform (3 x ml). The resin was sucked dry and the peptide extracted with i N aqueous acetic acid (4 x 30 ml). The combined extracts were lyophilized, redissolved in distilled water (7 ml) and relyophilized S86/04334 PCTiEP86/00012 77 to give 816 mg of the acetate salt of the crude peptide, Lys-Ser.

Analogously Lys-Val-Lys i8 obtained from tert.utoxycarbonyl--2-chlorbenzoxy-carbonyl-L-lysine-resin with S t-butoxycarbonyl-L-valine and t-butoxycarbonyl-E-benzoxycarbonyl- L-iysin e EXAMPLE 4 Pucification of Lvs-Ser( using carboxmethyl cellulose chromatography. The crude pept4de salt from Example 3 (2.5 g) was dissolved in distilled water (1 the pH adjusted to 5.0-6.5 (1 M, aqueots ammonia) and the solution applied to a column (2.5 x 30 cm) of Whatman CM-52 which had been equilibrated with 0.01 M ammonium acetate, pH 5.0-6.5. The column was washed copiously with the equilibration butlfer and then eluted with a linear gradient of 0.01 M ammonium acetate, pH 4.5-6.5 (350 ml) to 0.30 M ammonium acetate, pH (350 ml). The PH of the column and the starting buffer was adjusted to equal that of the peptide solution before loading. The fractions containing the pure product were combined and lyophilized to give SL-lysyl-L-serine as a colorless, hygroscopic solid.

.t Analogously Lys-Val-Lys is purified.

1 WO 86/04334 PCT/EP86/00012 78 EXAMPLE Purification of Lys-Ser using carboxymethyl Sephadex chromatography. Following the general procedure described in Example 4, the crude peptide from Example 2 (2 g) was dissolved in distilled water (1 the pH adjusted to 4.5-6.5 (1 M aqueous ammonia) and the solution applied to a column (2.5 x 30 cm) of CM-Sephadex (Pharmacia, Inc.) which had been equilibrated with 0.01 M .ammonium acetate, pH 4.5-6.5.

The column was then eluted with a linear' gradient of 0.01 M ammonium acetate, pH 4.5-6.5 (350 ml) to 0.30 M ammonium acetate, pH 6.5 (350 ml). The pH of the column and the starting buffer was adjusted to equal that of the peptide solution before loading. Fractions containing the pure product were combined and lyophilized to give L-lysyl-L-serine as a colorless, glassy, hygroscopic solid.

Analogously, Lys-Val-Lys is purified.

EXAMPLE 6 Purification of Lys-Ser usin reverse-phase chromatography on C-18 silica. The crude peptide from Example 1 (5.0 g) was dissolved in a minimum volume of distilled water (approximately 10 ml) and applied to a column (5 x 50 cm) of C-18 silica gel (Separation Technologies, Inc.) which had been previously equilibrated with aqueous hydrochloric acid. The column was eluted with the same solvent and the fractione containing the pure product combined and lyophilized to give L-lysyl-L-serine hydrochloiide salt as a colorless, hygroscopic, glassy solid.

Analogously, Lys-Val-Lys is purified.

a 6 PCT/EP86/00012 WO 86/04334 79 EXAMPLE 7 Purification of Lys-Ser using Sephadex G-10 chromatography.

The peptiae from Example 2 (1.0 g) was dissolved in a minimum volume of distilled water (5 ml) and applied to a column (2.5 x 100 cm) of Sephadex G-10 (Pharmacia) which had been equilibrated with 3% aqueous acetic acid. The column was eluted with the same solvent and the fractions which contained the pure product combined and lyophi7ized to give L-lysyl-L-serine acetate salt as a colorless, glassy, hygroscopic solid.

Analogously-, Lys-Val-Lys is purified.

EXAMPLE 8 Automated solid-phase synthesis of Lys-NH 2 on benzhydrylamine S resins. N-t-butyloxycarbonyl-e-benzyloxycarbonyl-L-lysine (2.31 g, 6.08 mmole) w"a coupled to preswollen benzhydrylamine resin crosslinked, substitution 0.76 meq. amine per gram) in dichloromethane ml) using a solution of dicyclohexyl:arbodiimide in dichloromethane (0.5 M, 12.2 ml; 2 equivalents) in an automated synthesizer, as described in Example 3. The mixture was stirred for 90 minutes, the resin thoroughly washed (see protocol in Example 3, steps 7-10),

S

20 transferred to a sintered glass funnel, washed successively with ethanol and dichlo,:omethane and dried under vacuum. Yield: 4.7 g.

The crude peptide, Lys-NH 2 was cleaved from the resin using liquid hydrogen fluoride, following the procedure described in Example 3. Yield: 693 mg.

Analogously, Lys-Val-Lys-NH 2 is prepared.

Peptide amides may be synthesized by an identical procedure, using 4-methylbenzhydrylamine reoin in place of benzhydrylamine resin.

WO 86/0433 PCT/EP86/00012 EXAMPLE 9 Synthes.s of No-acetyl-Lys-Ser (Ac-Lys-Ser) by acetylation of a peptide resin. The dipeptide was assembled by the solid-1-hase method, as described in Example 3, starting with 2.9 g of t-butyloxycarbonyl-0-benzyl-L-serine resin (subst-Itution 0.65 mmole/g) After cleavage of the N--terminal t-lautyioxycarbonyl group f rom the dipeptide-resin, it was acetylated using the following protocol: 1. Wash with dichloromethane (50 3 x 1 minute; ethanol ml), 2 x I minute; and dichloromethane ('50 ml), 3 x 1 minute.

2. Neutralize with triethylamine (10% in dichioromethane, ml), 1 x 1 minute and 1 x 5 minutes.

3. Acetylate by adding dichloromethane (40 ml), followed by acetic anhydride (1.9 ml pee mmole of peptide-rvalsin) and triethylamine L(2.8 ml per mmole of peptide-resin) for 20 minutes.

4. Repeat step 1.

The resin was collected on a sintered glass funnel, washed successively with ethanol and dichloromethane and dria~d under vacuum.

The peptide was cleaved from the resin using liquid hydrogen fluoride at 0 0 C, as described in Example 3 and purified by chromatography on C-18 silica, as described in Example 6 to give Nl~-acetyl- L-lysyl-L-serine acetate as a colorless, glassy hygroscopic solid.

Yield: 304.7 mg.

EXAMPLE Synthesis of D-Lysine N-ethylamide (D-Lys-NHCH 2

CR

3 via aminolvsi-s of an amino acid resin. No -t-Sutyloxycarbonyl-c-benzyloxycarbonyl-D-lysine- resin (substitution =0.67 mmole/g, 1.5 g) was S 6 vO 86/04334 PCT/EP86/00012 81 suspended in dimethylformamide (15 ml) and anhydrous ethylamine gas bubbled slowly through the solution until saturation was achieved and thie mixture stored in a stoppered flask for 24 hours at room temperature. The resin was removed by filtration, washed with dimethylformamide and the filtrate evaporated under reduced pressure. The residue was triturated with ether to give crude N-t-butyloxycarbonyl-c -benzyloxycarbonyl-D-lysine N-ethylamide as a white solid. Yield: 0.4 7.

The product was deprotected using liquid hydrogen fluoride ml) at 0OC in the presence of anisole (2 ml), as described in Example 3. Purification was accomplished by chromatography on Sephadex as described in Example 7 to give D-lysine N-ethylamide as a white, hygroscopic solid. Yield: 70 mg.

EXAMPLE 11 General method for the preparation of t-butyloxycarbonyl amino acid resins. Chloromethylated polystyrene-beads (Biobeads SX-1, Bio-Rad; 1% crosslinked; substitution 1.25 milliequivalents Cl/g) are added to a solution of the t-butyloxycarbonyl-amino acid deriva- S tive (1 equivalent) in dimethylsulfoxide (3 ml per gram of resin) in a round bottom flask, and the mixture treated with a solution of potassium t-butoxide (1 equivalent) in dimethylsulfoxide (1.5 ml per gram of resin; centrifuged to remove insolubles). The mixture is heated with agitation at 80 0 C using an oilbath for two hours and then allowed to stand at room temperature overnight. The resin is transferred to a sintered glass funnel, washed with dimethylsulfoxide WO 86/04334 PCT/EP86/00012 82 (3 times), alternately with methanol and dichloromethane (3 times each) and finally with methanol (2 times) and dried under vacuum.

Degree of substitution is calculated from the weight gain of the resin.

EXAMPLE 12 Acetate salts of the peptides listed in Table 6 were p:epared and purified under the protocols described in the foregoing examples, using the materials and protocols specified in the table. Peptide Rf and specific rotation data are given in Table 7.

EXAMPLE 13 Solution-phase'synthesis of Lys-Val-Lys acetate salt.

Tert-butyloxycarbonyl-L-valine (22.8 g, 0.105 moles) is dissolved in dry tetrahydrofuran (400 ml), the solution cooled to -150C and treated with N-methylmorpholine (11.0 ml, 0.1 mole), followed by isobutyl chloroformate (13.0 ml, 0.1 mole). The mixture is stirred at -150 for 10 minutes and then treated with a precooled colution of e-benzyloxycarbonyl-L-lysine benzyl ester hydrochloride (40.65 g, 0.1 mole) in tetrahydrofuran (200 ml) and dimethylformamide (50 ml). The reaction mixture is allowed to warm slowly to room temperature and stirred overnight. The solvent is evaporated under reduced pressure, the residue redissolved in ethyl acetate (2 1) and washed with 1 N hydrochloric acid (3 x 500 ml), 5% sodium chloride (3 x 500 ml), 5% sodium bicarbonate SWO 86/04334 PCT/EP86/00012 83 (3 x 500 ml) and finally with water (3 x 500 ml). The organic phase is dried over magnesium sulfate, concentrated under reduced pressure and crystallization of the product induced by addition of hexane to the cloud point. Yield: 46.7 g Tert-butyloxycarbonyl-L-valyl-c-benzyloxycarbonyl-Llysine benzyl ester from the previous step (46.7 g) is deprotected by dissolving in dichloromethane (250 ml) and treating with cold (OOC) trifluoroacetic acid (150 ml) for 30 minutes.

The solvent is evaporated under reduced pressure and the residue triturated several times with ether to give L-valyl-c-benzyloxycarbonyl-L-lysine benzyl ester trifluoroacetate salt in quantitative yield, which may be used without further purification.

N,N-bis-benzyloxycarbonyl-L-lysine (35.6 g, 0.086 mole) is dissolved in tetrahydrofuran (350 ml), the solution cooled to -15 0 C and treated with N-methylmorpholine (9.02 ml, 0.082 mole) followed by isobutyl chloroformate (10.64 ml, 0.082 mole). The mixture is stirred for 10 minutes and then treated with a precooled solution of L-valyl-e-benzyloxycarbonyl-L-lysine benzyl ester trifluotoacetate (46.4 g, 0.082 mole) in tetrahydrofuran (200 ml) and dimethylformamide (50 ml), followed by N-methylmorpholine (9.02 ml, 0.082 mole). The mixture is allowed to warm slowly to room temperature and stirred overnight. The product is worked up as described above to give N,N-bis-benzyloxycarbonyl-Llysyl-L-valyl-c-benzyloxycarbonyl-L-lysine benzyl ester. Yield: 49.0 g 7 i I- 141 WO! 86/04334 PCT/EP86/00012 84 The protected tripeptide (49.0 g) is dissolved in glacial acetic acid (1 1) and hydrogenated over palladium on carbon 10 g) overnight at atmospheric pressure. The catalyst is removed by filtration, the filtrate evaporated and the residue redissolved and lyophilized to give L-lysyl-L-valyl- L-lysine acetate salt in quantitative yield (37.0 g).

Purification may be achieved by the methods of Examples 4 through 7.

I

i.- 1 1 4 i W686/04334 W6 8604334PCT/EP86/0001I TABLE 6 Product Peptide S* Starting Resin Coupling Cycle Reagents P** D-Lys -NH 2 LysrSer -'Nf 2 D-Lys-Ser Desamino-Lys-Ser Orn-D-Ala Arg-K)-Ala His-Ser Lys-Cys Orn-Gly -Ly s-Ph e Lys -Asp Lys-Pro Ac-Lys-Ser

-NH

2 N-Me -L ys D-Se r Ala-Lys-Ser D-Ala-D-Lys-D-Ser Val-Lys-Ser fte-Lys-D-Ala L y s-Se r-Ty r Phe-Lys-Ser-Tyr 8 Boc-c-Cbz-D-Lys benzhydrylamine 8 Boc-OBzl-L-Ser benzhydrylamine 3 Boc-OBzl-L-Ser 3 Boc-OBzl-L-Ser 3 Boc-D-Ala 3 Boc-D-Ala 3 Boc-OBzl-L-Ser 3 Boc-6-MeCbz-Cys 3 Scc-Gly 3 Boc-L-Phe 3 Boc-a-Cbz-L-Asp 3 Boc-L-Pro 9,9 Boc-OBzl-L-Ser benzhydrylamine 3 Hoc-O8zl-L-Ser 3 Soc-O~zl-L-Ser 3 Boc-OBzl-D-Ser 3 Boc-OBzl-L 1 -Ser 3 BOC-D-Ala 3 Boc-Ofl-L-Tyr 3 Soc-O8zl-L-Tyr Boc-c -Cbz-L-Lys Boc-c -Cbz-D-Lys t-Soc-des-m-amino-Lys Boc-5 -Cbz-L-Orn Boc-N-tosyl-L-Arg Bo -i -o y -i Hoc-c -Cbz-L-Lys Hoc-d -Cbz-L-Orn Hoc-c -Cbz-L-Lys Hoc-c -Cbz-L-Lys Boc-e-Cbz-L-Lys Hoc-c -Cbz-L-Lys Hoc-NO -Mec -Cbz-L-Lys (threefold excess) Boc-e-Cbz-L-Lys; Boc-L-Ala Hoc-c -Cbz-D-Lys; Hoc-D-Ala Boc-c-Cbz-L-Lys; Boc-L-Val Boc-c -Cbz-L-Lys; Boc-L-Phe Boc-OBzl-L-Ser; Boc-e -Cbz-L-Lys Boc-Oftl-L-Ser; Boc-L-Phe 4,7 4,7 6 4,7 4,7 7 4,7 4,7 6 7 4,7 6 1) 6,7 6 7 7 6 2) q 6 1 /Table 6 continues an next page 7 WO 86/04334 PCT/EP86/000 12 Lys-Glu-Lys Lys-Gln-Lys Lys-Arg-Lys Lys-His-Lys Lys-D-Ala-Lys Lys -Le u-Lys -NE 2 Lys-Pro-Lys-NH 2 Lys-Tyr-Lys-NH 2 Lys-Ser-Arg 2m Lys-Thr-Arg Arg-GIly-Lys Arg-Ala-Lys Ala-Lys-Val-Lys-NH 2 Arg-Lys-Ser-Arg Ac-Asn-Lys-Ser-Arg 86 /Table 6, continued7 3 Soc-c-Cbz-L-Lys Boc-Y-'Sz1-L-Glu; Boc-c -Cbz-L-Lys 3 Soc-c-Cbz-L-Lys Boc-X-L-Gln; Soc-c -Cbz-L-Lys 3 Boc-c-Cbz-L-Lys Bo-gtslLAg Soc-c -Cbz-L-Lys 3 Soc-c -Cbz-L-Lys Bo-i-oslLHs; Boc-c-Cbz-L-Lys 3 Boc-c-Cbz-,-L-Lys Soc-D-AJla; Soc--e-Cbz-L-Lys 8 Boc-e-Cbz-L-Lys Boc-L-Leu; benzhydrylamine Soc-c -Cbz-L-LYS 8 Boc-e-Cbz-L-LVs Boc-L-Pro; berizhydryiamine Soc-E-Cbz-L-Lys 0 Boc-c-Cbt-L-Lys Boc-O~zl-L-Tyr; benzhydryiamine Soc---Cbz-L-Lys 3 Boc-Ng- Boc-OBzl-L-Se'r; tosyl-L-Arg Boc-c-Cbz-L-Lys 3 Boc-Ng- Soc-OBzl-L-Thr tosyl-L-Arg Soc-e -Cbz-L-Lys 3 Boc-e-Cbz-L-Lys Boc-Gly; Bo-gtslLAr S oc-c -Cbz-L-Lys Soc-L-Ala; 3 5O~N~ ~Boc-Ng-tos-*L-A; tosyl-L-Arg BO-gtslr-r 8 Boc-t-Cbz-L-Lys Soc-L-Val; berizhydvylainine Soc-c -Cbz-L-Lys; Soc-L-Ala 3 Boc-Ng-, Soc-OBzl-L-Ser; tosyl--L-Arg Soc- -Cbz-L-Lys; BO-gtsy--r 3,9 Boc-Ng- Boc-O~zl-L-Ser; tosyl-L-Arg Soc-c-Cbz-L-Lys; Boc-Xan-L-Asn" /Table 6 continues on next pagR7 7 7 7 7 41,7 7 7' 7 3 7 4,7 4,7 7 -7 w6O 86/04334 PCT/EP86/000 12 Gly-Asp-Lys 'er-Arg Asp- L ys-Ly s-Ar g Asp-Lys-Ile-Arg Gly-Lys-Val-Arg Asp-Orn-Ser-Arg Asp-Lys-Ser-His 3 Soc-Ngtosyl-L-Arg 3 Soc-Ngtosyl-L-Arg 3 Soc-Ngtosyl-L-Arg 3 Soc-Ngtosyl-L-Arg 3 Boc-Ngtosyl-L-Arg 3 Boc-Nim-, tosyl-L-His (Table 6 continued] Boc-O~zl-L-Ser; Boc-c -Cbz-L-Lys; Soc-B -Bzl-L-Asp; Soc-Gly Soc-c -Cbz-L-Lys; Boc-c-Cbz-L-Lys; Bq';--Dzl-L-Asp Boc-L-Ile; Soc-c -Cbz-L-Lys; Soc-sB zl-L-Asp Boc-L-Val; Soc-c -Cbz-L-Lys; Boc-Gly Boc-O~zl-L-Ser; Boc-s -Cbz-L-Orn Soc-sB zl-L-Asp Hoc-OBzl-L-Ser; Soc-c -Cbz-L-Lys; Soc-a -Bzl-L-Asp 7 7 7 61 7 7 S refers to the synthesis protocol'followed, as describ .ed in the Example number(s) given in thi's column.

refers to the purif ication protocol followed, as descrtbed in the Example number(s) given in this column. Where two protoco .s are listed, 11hese were utilized in the order listed.

1) 0-60% acetoni trule gradient in 0.5% aqueous HCl used for elution.

2) 0-60% acetonitrile gradient in 0.1 M ammonium acetate, pH 4.5, used for elution.

3) 0-30% acetonittile gradient in 0.51%0 aqueous HCI used for elution.

4) 0-50% acetonitrile gradient in 0.5% aqueoua HCl used for elution.

Note: The following abbreviations apply to Table 6: Socf tertbutyloxycarbonyl; Cbz, benzyloxycarbonyl; O13zlp O-benzyl; MeBzl, 4-methylbenzyl; Szl, benzyl; Me, methyl; Xan,, xanthydryl.

i 14111

~L~LII

WO 86/04334 PCT/EP86/00012 88 TABLE 7 PEPTIDE Rf AND SPECIFIC ROTATION

DATA

Product Peptide Lys-Ser Lys-NA2 D-Lys-NH 2 Lys-Ser-NH 2 D-Lys-Ser Desamino-Lys-Ser Orn-D-Ala Arg-D-Ala His-Ser Lys-Cys Orn-Gly Lys-Phe Lys-Asp Lys-Pto Ac-Lys-SerT-NH 2 N-Me-Lys-D-Ser Ala-Lys-Ser D-Ala-D-Lys-D-Ser Va-Ly-Se Phe-Lys-0-Ala Lys-Ser-Tyr Phe-Lys-Ser-Tyr R f 0.15 0.15 0.14 0.16 0.15 0.34 0.15 0.28 0.16 0.18, 0.16 0.37 0.20 0.14 0.26 0.13 0.15 0. 10 0.16 0.32 0.08 0.05 cd 2/ 20.42 (4.26) 16.21 (4.38) 13.39 (3.66) 20.32 (4.92) 42.54 (4.00) 2.36 (6.78) 64.36 (5.36) 56.15 (6.50) 31.44 (3.60) 9.70 (3.40) 44.20 (4.14) 1.46 (3.20) 24.11 (11.60) 55.60 (4.04) 35.50 (4.00) 51.23 (4.06) 17.25 (4.00) 17.88 (3.41) 0.61 (14.8) 16.67 (4.50) 18.78. (4.10) 7.01 (4.28) /Table 7 continues on next page/ 1/ TLC analysis on Silica Gel 60 plates (MERCK) in the solvent system n-butanol:acetic aci.d:water:ethyl acetate 2/ Specific rotation /L measured in water at the concentration (in mg/mi) indicated in parentheses.

Iv 86043 PCT/EP86/000 12 89 /Table 7, continued/ Lys-\Ial-Lys 0.10 4.78 (3.56) Lys-iVil-Lys-NH 2 0.09 -10.78 (4.64) Lys-Glu-Lys 0.09 1.84 (5.42) Lys-Gln-Lys 0.06 rid Lys-Arg-Lys 0.07 2.57 (15.8) Lys-His-Lys 0.03 9. 86 .(5.36) Lys-D-Ala-Lys 0.07 +45.03 (6.44) Lys-Leu-Lys-NH 2 '1.09 5.46 (4.60) Lys-Pro-Lys-NH 2 0.05 -41.67 (5.04) Lys-Tyr-Lys-NH 2 0.14 +30.88 (4.34) Lys-Ser-Arg 0.06 rid Lys-Thr-Arg 0.09 1.36 (5.88) Arg-Gly-Lys 0.08 +10.38 (5.20) Arg-Ala-Lys 0.10 4,59 (3.92) Arg-Asp-Arg 0.14 3.95 (1840) Ala-Lys-Val-Ly's-NH 2 0.07 -45.57 (4.06) Arg-Lys-Ser-Arg 0.01 nd Ad-Asr-Lys-Ser-Arg 0 .13 n d Gly-Asp-Lys-Ser-Arg 0.03 -32,50 (32.4) Asp-Lys-Lys-Arg 0.03 nd Asp-Lys-Ile-Arg 0.10 rid Gly-Lys-\Ial-Arg 0.12 rid Asp-.Orn-Ser-Arg 0.06 -20.49 (3.49) Asp-Lys-Ser-His 0.03 rid PCT/EP86/0O 12 WO 86/04334 In addition 'co the peptides mentioned above, the following preferred compounds can be prepared in analogy to the Examples: Ac-Asn-Lys-Val-Arg Des-e'.amino-Lys-Gln Ac-rLys-Ser-NH-CH 3 Des -amino- L ys-Gin- L ys Ala-Lys-D-Al& Des-J.-amino-Lys-Gly Ala-Lys-Ala-Lys Des-7.-amino-Lys-Gly-Lys Ala-Lys-Gin iDes-'z(-amino-Lys-His-Lys Ala-Lys-Oly-Lys Des-. ,-amino-Lys-HomoSer Ala-Lys-Phe iDes-. -amino-Lys-Lys-Lys Ala-Lys-Pro Des- -amino-Lys-Lys-Thr-Glu-Thr Ala-Lys-Sar IDes- k-amino-Lys-NH 2 Ala-D-Lys-D-Ser I ies--I-amino-Lys-Pro Ala-Lys-Val-Arg Des-Ck-amino-Lys-Ser-Tyl Ala-Lys-Thr,-Lys Des-V4-amino-Lys-Thr-Glu Ala-Urn-Ala Des-Y.k-amino-Lys-Thr-Glu-Thr Ala-Orn-D-Ala Des-'l-amino-Lys-Thr-Glu-Thr- Ala-Orn-Ser Gln-Glu-Lys Ala-D-Orn-Ser IDes-L-amiino-Lys-Thr-Lys Arg-Ala Des-)..-amino,ys-Val-Lys Arg-Gln Des t amino- Lys- Va 1- L ys-NH 2 Arg-Gly Gln-Qlu-Lys Arg-Pro Gin- Glu L ys-As Pr'o -L eu-Pro Arg-Ser Gln-Lys-Ser Arg-Ser-Arg GlU-Lys-Asn-Pro-L, u-Pro Arg-Ser=Lys GlU-Lys-Ser-Arg Arg-Thr-Lys C-Uu-Tht' Asp-Arg-Ser-Arg Glu-Thr-Gln-Glu-L.ys Asp-Lys-Ala-Arg His-Ala Asp-Lys-Ser-Arg-NH 2 Hia-Gly HomoLys-Gly Des-&-amino-Lys-Ala HomoLys-Srir Des 4-amino-Ls,,s-Ala-Lys Leu-Lys-Ly's-Thr-IGlu-Thr Des-1,.amino-Lys-'Arg-Lys Lys-Ala Deb-i.-amino-Lye-Asp-Lys Lys-D-Ale Des _-amino-I.,ys-Cy a Lys-Ala-Lys v686/04334 wd 8604334PCT/EP86/000 12 Lys-Ala-Tyr Phe-Lys-Ser Lys-Asp-LYs Sar.,Lys-Ser Lys-Gln-Lys-NH 2 Sa D-Lys-Ser Lys-Glu-Lys-Nrii 2 Thr-Gln-Glu-Lys Lys-Gly Thr-Gln-Giu-Lys-Asn-Pro-Leu-Pro Lys-Gly-Lys Thr-Glu-Thr Lys-Gly-Lys-NH 2 Thr-Glu-Thr-Gln-Glu-Lys Lys-Gly-Tyr Va.-Lys-Thr-Arg.

Lys-Lys-Lys Lys-Lys-Thr Lys-Lys-Thr-G1u-Thr Uys.-Phe-Lys-NH, 2 Lys-.Ser-NH-CH D-Ly-Ser-NH 2 D-Lys-Ser-NH-CH 3 lys-Ser-NH-r 2 D-Lys-Ser-i\, -C 2 H Lys-Thr-Glu Lys-Thr-Glu'-Thr L~ys-Thr-Glu-Thr-Gln-Glu-Lys Lys-Thr-'.,ys N-Me-Lys-Ser N-'Me-Lys-Ser-NH 2 N-Me-Lys-Ser-NH-CH 3 N-Me-Lys-Ser-NH-C 2 H Or n Se r D-Orn-Ser Orn-Ser-NH 2 D-Orn-Ser-NH 2 DOrn-Ser-NH-CH 3 DOrn-Ser-H CH D -Or n-Set'-NN~- C. H Phe-Lys-Gly

WW

WO 86/043:34 PCT/EP86/00012 92 in the syntheses described the foregoing examples, the solvents and reagents were invariabLy of the highest, commercially-available grade and were used without purification, except in the following cAses: Triethylamine was dried over solid potassium hydroxide, decanted and distilled from ninhydrin (1 g per liter of triethylamine) at at~mospheric pressure.

Trifluoroacetic Acid was distilled from phosphorus pentoxide at atmospheria pressure.

Dicyclohexylamine was dried over solid potassium hydroxide and distilled under vacuum.

Dioxane was dried over metallic sodium,, pieces (freshly *cut) and distilled at atmospheric pressure.

Dimethylformaraide was dried over solid potassium hydroxide, decanted and distilled from ninhydrin (1 g per liter of solvent) under vacuum.

Pyridine was dried over potassium hydroxide, decanted and distilled from ninhydrin (1 g per liter of solvent) at atmospheric pressure.

EXAMPE 2x4 Methods for assessing Purity of intermediates and inal products. The purtty of intermediates, c~olumn fractions (from purifications) and final products was assessed by a cqimbination of techniques. Thin layer thromatography (TLC) was car;-ied outJ on glass-backed Silica Gel 60 plates (Merck) using the ftollowing solvent systems,.

1. nTbutanol:acetic acid:water:pyridine (30:6:20:24)f* PCT/EP86/00012 OWO 86/04334 9 93 2. n-butanol:acetic acid:water:ethyl acetate and 3. isopropanol:ammonia Products were visualized by spraying the plates with ninhydrin solution in ethanol) or chlorine peptide spray solution of t-butyl hypochlorite in cyclohexane, followed by a 1% solution of o-toluidine in 10% acetic acid).

High performance liquid chromatography (HPLC) was carried out on C-18 reverse-phase columns (5 um particle size, 4.6 mm i=6. x 25 cm) using gradients of acetonitrile in 5 mM aqueous hexanesulfonic acid or octanesulfonic .cid, Flow-cates were typically 1 ml/min., chromatograms were monitored at 215 nm and 10-20 ugloads of samples were injected.

High voltage electrophoresis was carried out in pyridine:acetic acid:water (10:0.4:90) buffers on Whatman 3MM paper at 2 kV. Products were visualized by spraying the chromatograms with ninhydrin solution in ethanol).

Final, purified peptide products were packaged under sterile conditions in vials containing 1 ml of 0.01M phosphate buffer, pH 7.4, per milligram of peptide and lyophilized to a dry powder for storage.

EXAMPLE Murine Mixed Lymphocyte Response Assay Procedure. The murine MLR assay procedure is described in Dutton, J. Exp.

Med., 123:655-671 (1966). The spleen is removed from mice of two different strains, and the spleen cells from each strain are disbursed and separately suspended in human serum albumin in RPMI- 1640 RSA-RMPI, Irvine Scientific). The cell suspensions are WO 86/04334 PC'TEP86/00012 94 then eantrifuged at 400 G for ten minutes at 15°C, and the cell pellets resuspended in 2% HSA-RPMI.to a total volume of 50 ml.

The cell concentration is ascertained using, a hemacytometer. Cell viability, as measured by trypan blue dye exclusion, should be at least 95%. A stimulator cell suspension is prepared by incubating the cells of one strain with 25 ug mitomycin C per mi for 30-60 minutes at 37°C or irradiation with a total of 3200 roentgens in order to inhibit DNA synthesis. After washing the stimulator cells three times with 2% HSA-RPMI, both the stimulator cells and the untreated responder cells are suspended in KC 2000 (KC Biologicals) to a concentration of 4 x 106 cells/ml.

Equal portions of each suspension are then combined,, and a control group is prepared in a microtiter plate well by transferring 0.1 ml aliquots each of the cell mixture and KC 2000. An experimental group is prepared by transferring to a microtiter plate well 0.1 ml of the cell mixture and 0.1 ml of the test peptide diluted to twice the experimental concentration. The microtiter plates are incubated in a humidified 37 0 C incubator with an atmosphere of 5% CO 2 for seven days, whereupon 1 UCi 3 H-thyridine 20 is added in 0.025 ml/well. The control and experimental groups are incubated for six hours and then harvested using a 12-channel SCATRON cell harvester or the equivalent. After the filter pods have air dried, the radioactivity count due to incorpeoated 3 H-thymidine is ascertained in a LKB minivial scintillation counter using 3 ml Cytoscint (Westchem). The amount of cell-associated radioactivity is used to estimate the degree to which the responding cells in the experimental and control groups are stimulated to proliferate in the presence and in the absence of the subject peptide.

W 86/04334 PCT/EP86/00012 EXAMPLE 16 Human Mixed Lymphocyte Response Assay Procedure. The human MLR assay is discussed in "Lymphocyte Transformation in Cultures of Mixed Leukocytes," Lancet 1:1184-1186 (1965), and in S 5 Walthe, W.I. et al. (Weir, Handbook of Experimental Immunology (3d pp. 26.1-26.10, Blackwell Scientific Publications (London 1978). In this assay, venous blood is extracted from two unrelated human subjects (A and B) in sterile syringes containing 0.15 ml heparin (10,000 units/ml) per 60 cc blood.

The mononuclear cells are isolated by centrifuging 40 ml of a 1:1 mixture of blood and sterile RPMI-1640 (Irvine Scientific), underlaid with 8 ml Ficoll-Paque (Pharmacia Fine Chemicals), for minutes at 400 G and 150C. The plasma is discarded and the mononuclear cell band adjusted to 40 ml with 2% human serum albumin in RPMI-1640 HSA-RPMI, Irvine Scientific). The cell mixture is centrifuged for 8 minutes at 40 G and 15C, residual plasma discarded, and the cell band rediluted and recentrifuged as immediately above. The cell pellet is resuspended in 10.0 ml KC 2000 (KC Biologicals), and the mononuclear cells counted using Sa Coulter counter or the equivalent. Stimulator cells and are prepared by treating 20 X 106 mononuclear cells with irradiation or sitomycin C as described above in the murine MLR assay procedure. Responder cells (A and B) are prepared by washing the untreated cells once with 2% HSA-RPMI. Both responder and stimulator cells are adjusted to a final concentration of 2 X 106 mononuclear cells/ml in KC 2000. In a typical one way human MLR assay, experimental and control groups are assayed by incubating equal-volume mixtures of, responder cells A and stimulator cells B* for seven days with and without the subject mLI WO 86/04334 PCT/EP86/00012 96 peptide, as described in the murine MLR procedure. 3 H-thymidine is then added to each sample, followed by incubation for six hours, harvesting, and counting of the incorporated labelled DNA in the newly replicated cells as in the murine assay.

EXAMPLE 17 Antigen-Specific T Cell Proliferation Assay Procedure.

The antigen-specific T-cell proliferation assay consists of injecting a live mouse with a specific antigen for a period sufficient to allow sensitization to the antigen, culturing the sensitized lymph node ,cells,. and then reexposing the lymph node cells to the antigen after treatment with the subject peptide.

Following an appropriate incubation period, the amount of immunoresponsive T-cell proliferation is measured using labelled-DNA ,uptake methods, tritiated thymidine incorporation, thus providing a measure of the immunoregulatory activity of the subject peptide with respect to a specific antigen. The data in Table 2 reflect peptide-induced inhibition of T-cell proliferation in response to the antigen ovalbumin. Similar assays may be performed using such antigens as bovine serum albumin, human IgG, sheep, chicken or bovine erythrocytes, or haptens such as dinitrophenol.

The antigen-specific T cell proliferation assay is described in "Antigen Induced Proliferation Assay for Mouse T- Lymphocyte Response to Monovalent Antigens," Eur. J. Immuno.

8:112-118 (1978). A mouse is injected subcutaneously at the base i F~ ~Uc W 'W686/04334 PCT/EP86/00012 97 of the tail with 0.1 mg ovalbumin in 0.05 ml Complete Freund's Adjuvant. After seven days, the mouse is sacrificed and its superficial inguinal lymph nodes removed aseptically. Excess fat is removed, and the lymph node cells are dispersed into 15 ml of 2% human serum albumin (HSA) in phosphate-buffered saline (PBS) by, gentle teasi;g between the frosted surfaces of two sterile microscope slides. The dispersed single cells are then gently resuspended three to five times using a sterile plastic pipet, transferred to a sterile centrifuge tube, and the supernatent transferred to a new centrifuge tube, free of settled debris, after three minutes. The single cell suspension is centrifuged for ten minutes at 400 G and 15"C, and the pellet resuspended and tested for cell viability by measuring the exclusion of trypan blue dye solution, 0.020 ml) from cells of the cell suspension (0.100 ml). The cells should measure at least about 95% viable. The cells are then washed twice in 2% HSA-PBS and resuspended at approximately 8 x 106 lymph node cells/ml in Complete Click's Medium. The cell count is determined and the lymph node cell solution diluted to a final concentration of 4 x 106 cells/ml with Complete Click's Medium. 0.1 ml aliquots of the culture suspension are transferred into microtiter plate wells, and 0.1 ml of the subject peptide in Complete Click's Medium in the concentrations specified in Table 2 is added to the experimental groups. Control groups receive Complete Click's Medium. After at least 15 minutes, 100 ug/ml of ovalbumin is added to groups with and without the subject peptide. Control groups are prepared without antigen and with 100 ug/ml of anti- I WO 86/04334 PCT/EP86/00012 98 gens unrelated to ovalbumin human IgG) or with mitogens such as concanavalin A, phytohemagglutinen, and polkweed mitogen.

The plates are incubated at 37 0 C and 5% CO 2 On day four to day five, 1 uCi of 3 H-thymidine is added in 0.025 ml/well, and the plates are incubated for 24 hours before harvesting and counting as described with respect to the murine MLR assay procedure.

EXAMPLE 18 Leukemia and Tumor Cell Proliferation Assay. Mouse leukemia/lymphoma and human tumor cell lines are available from, American Type Culture Collection, i2301 Parklawn Drive, Rockville MD 20852-.'776. The individual cells are gently and sterilely dispersed to suspension in PBS. Cell viability is ascertained using the trypan blue dye exclusion method described in Example 16, and should be at least about 85%. The cells are washed 2-3 times in PBS and then twice in RPMI 1640. The cells are then resuspended in a sufficient quantity of approximately 10% FCS-RPMI or HSA-RPMI to yield a final cell concentration of 1 x 106 cells/ml. 0.1 ml aliquots of the tumor cell suspension are transferred to microtiter plate wells. 0.1 ml aliquots of appropriately-diluted peptide in RPMI 1640 are added as indicated in Tables 4 and 5. After incubation for 48 hours at 370'C and

CO

2 the cells are pulsed for six hours with 1 uCi 3 H-thymidine, harvested, and counted as described in the foregoing examples.

General procedures relating to the measurement of neoplastic cell proliferation may be found in Sample et al., Clin. Exp. Immunol. 9:419 (1971) and in Shellekens et al., Clin.

Exp. Immunol. 3:571 (1968).

Claims (14)

1. An immunoregulatory compound selected from the following formulas A-X-C A-X-B--Y-C iiIa lb wherein X and Y are each a residue of an amino acid with a pos itively charged side chain or an amino acid derivative with V o a positively charged side chain; and AB,C,X and Y are selected such that said compound has I any one of the following formulas as hereinbefore defined; from formula Ia: Iamg, Iamh, Iami, Iamj, Iamk, Iaml, Iamm, Iamn, ao lamp, Iamg, :amr, Iams, Iamat, Iamu, Iamv, Iamw, Iamx, 0000lamy, Iamz, lao, lap, laq, Iar, Ias, Iat; or from formula Ib: V. Ibo, Ibp, Ibq, Ibr, Ibs, Ibt, Ibu, Ibv, Ibw, Ibzc, Ibzf, Ibzg, Ibzh, Ibzi, Ibzj, Ibzk, Ibzl, Ibzm Ibzn, Ibzo.

2. A peptide selected from the group consisting of Ala-.Lys-Ser, Ala-D-Lys-D-Ser, Arg-Asp-Av:g, Asp-Lys-Ser, *aso: Des-m-amino-Lys-Ala-Lys, Des-cx-amino-Lys-Arg-Lys, Des -o-amino-Lys-Asp-Lys, Des -(x-amiiflO-LyS -Cys, Des -cc-dtmino-Lys--Gln, Des-oa-amino-Lys'*Gln-Lys, Des A~iino-T~y§-Gl1y -Lys, Des-c-amiAio--Lys-Hi s-Lys, Des arti no., Lys -Homo Se r, Des-cx-amino--Lys-Lys-Lys, Des -c-ami no-Lyz-Lys -Thr-G lu-Thr, Des-%-amino-Lys -Pre, -100- Des-ca-amino-Lys-Ser, Des-cm-amino-Lys-Ser-Tyr, Des-cm-amino-Lys-Thi,-Clu, Des-ca-amini-Lys-Thr-Glu-Thr, Des-cm-amino-Lys-Thr-Glu-Thr-Gln-Glu-Lys, D es-a~-amino-Lys-Thr-Lys, Des-a~-aminoLys-lVal-Lys, Des-om-amino-Lys-Val-Lys-NH 2 Gln-Glu--Lys, Glu-Lys--Asn- Pro-Leu-Pro, Glu-Thr-Gln-Glu-Lys, Gly-Lys-Ser, HornoLys-Gly, 1HomoLys-Ser, Leu-Lys-Lys-Thr-Glu-Thr, Lys-Ala-Tyr, Lys-Arg-Lys, Lys-Cys, Lys-Gin, Lys-Gin-Lys, Lys-Gly-Tyr, Lys-His-Lys, Lys-HomoSer, Lys-Lys-Thr-Glu-Thr, Lys-Thr-Glu, Lys-Thr-Glu-Thr, Lys-Thr-Lys, Lys-Val-Lys, Lys-Val-Lys-NH 2 Orn-Ala, Thr-Gln--Glu-Lys, Thr-Gln-Glu-Lys--Asn-Pro-Leu-Pro, Thr-Glu-Thr, [1 and Val-Lys-Ser.

3. A peptide selected from the group consisting of V Ac-Lyf--Ser, Ac-Lys-Ser-Lys, Ac-Lys-Ser-NH,,, Ala-Lys-Gly, D-Ala-D-Ly8-D-Ser, Ala-Lys--Ser-Arg, Ala-Lys-Val-Lys-NH 2 Arg-Ala-Lys, Arg-Gly-Lys, Arg-Lys-Ser-Arg, Arg-Sar, Arg-Thr, Asn-Lys-Ser-Arg, Asp-Hi -Ser-Arg, Asp-Lys--Ile-Arg, Asp-Lys-Lys-Arg, Asp-Lys-Ser-His, Asp-Orn--Ser-Arg, Gly-Asp-Lys-Ser-Arg, G? r-Lys-Val-Arg, Lys-D-Ala-Lys, Lys-As-p, Lys-Glu-Lys, Lys-Leu, Lys-Tbeu-LIys-NH 2 D-Lys-NH4 2 4 D-Lys-NHCH 2 CH 3 ,F Lyrs-D-Phe, D-tys-4Ser, LsSrAa 4 Lys-Ser-Arg, Lys-Ser-rNH, Lys-Thr-Arg, Lys-Tyr-LyS-NH 2 Lys-D-Val-Lys-rH 2 N-Me-.Lys--D-Ser, Orn-D-Ala, Phe-Lys-D-Ala, Phe-Lys-Sar, Phe-Lys--Ser-Tyr, Sar-D-Lys-Ser, Ser-Lys-Va.l-Arg, Tyr-Lys-Gly and Val-Lys-Val-Lys-NH 2

4. A process for the preparation of a peptide of any one of the preceding claims characterised in that such peptide is VJ i 6e/AC 0U 101 liberated from its corresponding functional derivative which may be bound by a covalent bond to a solid resin by treatment under acidic or basic conditions, and, if desired, the peptide thus obtained is esterified, amidated and/or acylated to yield a corresponding ester, amide, N- and/or O-acyl derivative or is transformed into one of its pharmacologically acceptable salts by treatment with ,1 base or an acid.

A pharmaceutical preparation comprising a peptide of any one of claims 1 to 3 and at least one pharmaceutically acceptable carrier.

6. A pharmaceutical preparation of claim 5 for suppressing autoimmune disease responses.

7. A pharmaceutical preparation of claim 5 for suppressing organ transplantation rejection responses.

8. A pharmaceutical preparation of claim 5 for suppressing neoplastic cell growth.

9. A pharmaceutical preparation of claim 5 for suppressing S T-lymphocyte proliferation in mixed lymphocyte response cultures or in antigen-specific T-lymphocyte proliferation assay cultures. i

10. The method for suppressing immume system responses in a subject which comprises administering to said subject an effective amount of an immunoregulatory compound according to any one of claims 1 to 3.

11. The method according to claim 10 for suppressing autoimmune disease responses.

12. The method according to claim 10 for suppressing organ A O2! 6 e/AC 1 1 4 I I .4 4 S i 4 4 54 5 *4 S :S 102 transplantation rejection responses.

13. The method According to claim 10 for suppressing neoplastic cell grcwth.

14. An immunoregulatory compound according to any one of claims 1 to 3, substantially as herein described with reference to any one of the Examples describing said compound. DATED this 18th day of July, 1990. IMMUNETECH PHARMACEUTICALS INC. By Its Patent Attorneys ARTHUR S. CAVE CO. L,