CA1197800A - Immune modulator peptides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A class of 23 or 24 amino acid peptides available by chemical synthesis or by enzymatic and chemical cleavage of IgG is described. The compounds are useful in modulating the immune response.

Description

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~-5 854 -1-IMMUNE MODULATOR PEPTIDES
This invention concerns a class of poly-peptides that are useful in modulating the immune response.
Immunoglobulins are proteins produced by plasma cells as one of the latter in a complex sequ~nce of events initiated by antigen challenge of the host.
The plasma cells, as another part of this sequence, are derived from B lymphocytes that have become activated by a mechanism or series of mechanisms that, to date, are not fully understood.
There are five classes of recognized immuno-globulins: IgG, IgA, IgM, IgD, and IgE. Of these, IgG
represents the major portion of circulating immuno-globulins.
The immunoglobulin protein molecule iscomposad of four interconnected polypeptide chains, two of which are termed "light" chains and two "heavy"
ch~;ns. Under this arrangement, the Ig molecule is divided into two identical portions, each of which comprises a light and a heavy chain linked by disulfide bridges formed from cysteine residues. The two result-ing portions, in turn, are linked by disulfide bridges.
Each of the four chains is composed generally o two portions, a variable region (VL and VH) extend-ing from the amino-tPrm; nal and having highly variable amino acid sequences, and a constant region (CL and CH) extPn~; ng from the carboxy terminal and having generally constant amino acid sequences. Each of these b~

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regions represents a discrete portion of the Ig mole-cule. The CH region is further subdivided into three domains designated CHl, CH2, and CH3 on the basis of constant homology regions.
It has been known for a number o years that the Ig molecule can be enzymatically cleaved into discrete fragments. The properties of these fragments then have been determined using a variety of biological assay systems.
Using papain, the IgG molecule can be cleaved, producing an "Fc" fragment and tw~ "Fab" fragments.
The Fc fragment represents the C-terminal portions of the two heavy chains joined by a disulfide bridge, and the Fab fragments are composed of the N-terminal por-tion of the heavy chain and the entire light chain joined by a disulfide bridge.
Pepsin and plasmin cleavages of the IgG
molecule occur at sites closer to the heavy chain C-terminal and downstream of the disulfide bridge that joins the two heavy ch~ins. The pepsin product, termed the "pFc"' ~ragment, and the plasmin product, thus represent C-terminal portions of the Fc fragment (CH3 domain of IgG).
It has been recognized that certain biologi-cal activities reside in the Fc and pFc' fragments.Thus, mouse spleen B lymphocytes are induced to pro-lifer~te in the presence of papain-derived Fc fragments, ~M. A. Berman and W. O~ Weigle, J. Exp. Med. 146, 241 (1~77)]. Furthermore, it has been observed that the proliferative response is dependent upon the presence ~7~0 of macrophages. It appears that macrophages enzymati-cally cleave the Fc fragment to a 14,000 MW subfrag-ment, and the latter stimulates B cell proliferation [E. L. Morgan and W. O. Weigle, J. Exp. Med. 150, 256 (1979); and E. L. Morgan and ~. O. Weigle, J Exp. Med.
51, 1 (1979)].
Subsequently, it was demonstrated that the Fc fragment has the ability in the presence of both macro-phage and T cells to induce a polyclonal antibody response in ~ouse spleen cells [E. L. Morgan and W. O.
Weigle, J. Immun. 124, 1330 (1980)].
Correspondingly, it was also demonstrated that the shorter fragment produced by plasmin digestion of IgG is active in producing a polyclonal antibody response [E. L. Morgan and W. O. Weigle, J. Supra-~molecular Structure 14, 201 (1980)].
A class of small peptides now has been dis-covered. These peptides are useful in modulating the immune response and are available either synthetically using readily available peptide synthesis methods or by en~ymatic and chemical cleavage of the IgG molecule.
When produced by cleavage methodology, the IgG first is digested with plasmin after which the resulting segments ara treated with cyanogen bromide. The resulting active fxagment comprises 23 amino acids defined by residues 335-357 of the IgG molecule to which is attached, at the C-terminal, homoserine (Hse) resulting from the methionine (residue 358), the site of the CNBr cleavage.
For an illustration of these structures, see, for X-5854 ~4~

example the sequence described in G. M. Edelman et al., Proc. Nat'l Acad. Sci. USA 63/ 78 (1969).
As noted, the peptides of formula I below are available synthetically. When so produced, the C-terminal homoserine may, if desired, be omitted.
It surprisingly has been discovered thatrel~tively small peptides as defined by formula I
exhibit a potentiation of the immune response in terms of their ability to activate T cells and natural killer cells, to initiate and/or promote B-cell differentation le~ding to antibody production, and to regulate an existing immune response. These polypeptides are quite selective in their action in that, unlike the Fc fra~-ment described above, these compounds do not induce a lS significant B cell proliferation.
Therefore, this invention concerns a class of compounds having the formula deined by a 23 amino acid sequence represented by residue 335-357 of an I~G
molecule to which is bonded, by amide formation, at the carboxyl terminal, homoserine or the lactone produced by dehydration of homoserine.
Preferred compounds of this class are those o Eormula I below having the structure H-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Glu-Glu-Hse in which Hse is homoserine or homoserine lactone. These compounds are preferably prepared by en7ymatic and chemical cleavage of the IgG molecule.

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~-5854 -5-This invention concerns a class of compounds of formula I having the structure R-Thr-a-b-Lys-d-e-f-g-h-j-k-l-m-n-p-q-t-u-Pro-v-w-x-y-(z)r-Rl in which each amino acid residue has an L-configuration;
R is hydrogen or Cl-C3 alkyl;
Rl is OH or NR3R4 in which R3 and R4 inde penden Lly are hydrogen or Cl-C3 alkyl;
a is Ile or Leu;
b is Ser or Ala;
d is Ala, Thr, Pro, Val, or Ser;
e is Lys, Arg, Thr, or Gly;
lS` f is Gly, Val, or Asn;
g is Gln, Lys, Ser, Glu, Pro, Ala, Asn, or Thr;
h is Pro, Val, Thr, or Phe;
j is Arg, Leu, Pro, or Phe;
- k is Glu~ Ala, Ile, Met, or Pro;
1 is Pro, Lys, or Gln;
m is Gln, Glu, or Val;
n is Val or His;
p is Tyr, His, or Leu;
q is Thr, Val, or l.eu;
t is Leu, Ile, Met, or Pro;
u is Pro or Gly;
v is Ser or Pro;
w is Arg, Gln, Glu, or Ser;
x is Glu, Asp, Gln, or Asn;
y is Glu, Gln, Gly, or Leu;
z is Hse; and r is 0 or 1.

~7~0~

X-585~ -6-A preferred subclass is represented by those compounds of formula I in which a is Ile;
b is Ser;
d is Ala, Thr, Pro, or Ser;
e is Lys or Arg;
f is Gly;
g is Gln, Lys, Ser, or Pro;
h is Pro or Val;
j is Arg or Leu;
k is Glu, Ala, Ilè, or Met;
1 is Pro;
m is Gln or Glu;
n is Val;
lS p is Tyr;
q is Thr, Val, or Leu;
t is Leu, Ile, or Met;
u is Pro or Gly;
v is Ser or Pro;
w is Arg;
x is Glu or Asp; and y is Glu or Gln.
A more preferred subclass of formula I is re~presented by those compounds in which a is Ile;
b is Ser;
d is Ala, Thr, or Ser;
e is Lys or Arg;
f is Gly;

X-5854 -7~

g is Gln;
h is Pro;
j is Arg;
k i s Glu;
1 is Pro;
m is Gln;
n is Val;
p is Tyr;
q is Thr;
t is Leu;
u is Pro or Gly;
v is Ser or Pro;
w is Arg;
x is Glu or Asp; and y is Glu or Gln. ~
A further more preferred subclass of formula I
is represented by those compounds in which a is Ile;
b is Ser;
d is Ala or Thr;
e is Lys;
f is Gly;
g is Gln;

2~ h is Pro;
j is Arg;
k is Glu;
1 iS Pro;
m is Glu or Gln;
n is Val;
p is Tyr;

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x-sas4 q is Thr;
t is Leu;
u is Pro;
v is Ser;
w is Arg or Gln;
x is Glu, Asp, or Gln; and y is Glu or Gly.
Examples of preferred amino acid sequences of fo~mula I are:
R-Thr-Ile-Sar-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Glu-Glu-Rl;
R-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Asp-Glu-Rl;
R-Thr-Ile-Ser-Lys-Thr-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-PrQ-ser-Arg-Glu-Glu-Rl;
R-Thr-Ile-Ser-Lys-Thr-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Glu-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Glu-Glu-Rl;
R-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Gln-Glu-Glu-Rl;
R-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Pro-Pro-Arg-Ile-Pro-m-Val-Tyr-Leu-Leu-Pro-Pro-Pro-Arg-x-y-R1;
R-Thr-Ile-Ser-Lys-Thr-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-~-Gly-Rl;
R-Thr~Ile-Ala-Lys-Val-Thr-Val-Asn-Thr-Phe-Pro-Pro-Gln-Val-His-Leu-Leu-Pro-Pro-Pro-ser-Glu-Glu-Rl;
and R-Thr-Leu-Ser-Lys-Ser-Gly-Asn-Thr-Phe-Arg-Pro-Gln-Val-His-Leu-Leu-Pro-Pro-Pro-Ser-w-x-Leu-Rl.

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The compounds of formula I where r is 1 can be prepared by en~ymatic and chemical cleavage of IgG. The IgG, obtained by routine methodology, can be purified by ammonium sulfate fractionation followed by DEAE cellulose chromatography using 0.OlM phosphate buffer, pH8, as eluant.
The recovered IgG then is digested with plasmin generally for 20 30 hours at an elevated tem-perature of about 37C. The active fragment of the resulting digested IgG can be separated on'Sepha~ex G-100 using O.OlM phosphate bu~er, pH7, as eluant.
The active portion then is treated with CNBr according to recognized procedures. These generally involve dissolving the product recovered from plasmin digestion in 70-90% formic acid containing an excess of CNBr. Cleavage is allowed to proceed at room tempera-ture over an extended period ~about 24 hours). The desired peptide product, a sequence comprising residues 335-358 of the-IgG, molecule, in which residue 358 2Q (me~hionine) is changed to homoserine or homoserine lactone by reason of the CNBr clea~age reaction, can be recovered by routine chromatographic separation, in-cluding molecular exclusion high performance liquid chromatography ~HPCL) and reverse phase HPLC. The resultin~ seyuence, comprising residues 335-358 as modified by CNBr cleavage, represents compounds of formula I where r is 1.
The compounds of formula I also can be prepared by any of a variety of recognized peptide syn~hesis techniques including classical (solution) Trademark for a highly cross-linked dextran, in the form of a Powder composed of macroscopic beads, which contains functional ionic groups attached to the glucose units of the polysaccharide chains by ether linkages.

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X-5854 -lO-methods, solid phase me~hods, and the more recently available recombinant DNA methods.
A ~referred method of preparation of the compounds of formula I is by the solid phase technique in which the amino acid sequence is constructed sequen-tially from an initial, insoluble, resin-supported C-terminal amino acid. Techniques for the solid phase method are described by J. Stewart et al., Solid _hase Peptide Synthesis, Freeman and Co., San Francisco, 1969.
In general, in the solid phase method, the amino acid corresponding to the C-terminal amino acid residue of the desired peptide is anchored to an in-soluble resin support, and the peptide chain then is formed beginnin~ at the resin-supported C-terminal amino acid. Individual amino acids are introduced sequentially until the desired amino acid sequence is obtained. Alternatively, small peptide fragments can be prepared and introduced into the peptide chain in the desired order. The peptide chain remains attached to the resin throughout synthesis, and, upon completion of the chain, the peptide is cleaved from the resin, The peptide chain is attached to the poly-styrene resin by means of an ester linkage formed between the carboxyl group of the C-terminal moiety and one of the methylene groups present on the resin matrix as sites for such attachment. The polystyrene xesin is a styrene polymer which is cross-linked by the addition o about 0.5 to a~out 3~ divinylbenzene and 7~

X-585~ -11-which is chloromethylated or hydroxymethylated toprovide sites for ester formation. An example of a hydroxymethylated resin i5 described by ~o~nszky et al., Chem. Ind. (London) 38, 1597-98 (1966). A chloro-mechylated polystyrene resin is commercially availablefrom Lab System, Inc., San Mateo, California. The res~n is also described by Stewart et al., Solid Phase Peptide Synthesis, Freeman and Co., San ~rancisco, California, pp. 1-6.
The amino acids are coupled using techniques well-known in the art for the formation of peptide bonds. One method involves converting the amino àcid to a derivative that will render the carboxyl group more susceptible to reaction with the free N-terminal amino group of the peptide fragment. For example, the amino acid can be converted to a mixed anhydride ~y reaction of a protected amino acid with ethyl chloro-formate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroormate, pivaloyl chloride, or similar acid chlorides. Alternatively, the amino acid can be converted to an active ester such as a 2,4,5-trichloro-phenyl ester, a pentachlorophenyl ester, a p-nitro-phenyl ester, an ester formed from N-hydroxysuccinimide, or an ester ~ormed rom l-hydroxybenzotriazole.
Another coupling method involves use of a suitable coupling agent, such as N,N'-dicyclohexyl-carbodiimide (D~C) or N,N'-diisopropylcarbodiimide (DIC). Other appropriate coupling agents will be apparent to those skilled in the art. [See Schroder ~L~97~

and Lubke, The Peptides, Academic Press, lg65, Chapter III.]
It should be recognized that the a-amino group of each amino acid employed in the peptide synthesis must be protected during the coupling xeaction to prevent side reactions involving the reactive a-amino function. It ~hould also be recognized that certain amino acids contain reactive side-chain functional groups (e~g. sulfhydryl, ~-amino, carboxyl, and hydroxyl), and that such functional groups must also be protected both during the initial and subsequent coupling steps.
Suitable protec~ing groups are known in the art ~See for example, Protective Groups In Organic Chemistr~, M.
McOmie, Editor, Plenum Press, N.Y., 1973.1 In selecting a particular protecting group, certain conditions must be observed. An a-amino protecting group (1) must be stable, (2) must render the a-amino ~unction inert under the conditions employed in the coupling reaction, and (3) must be readily removable after the coupling reaction under conditions that will not remove side chain protecting groups and will not alter the structure of the peptide fragment.
A side chain pro-tecting group ~1) must render the side chain functional group inert under the conditions employed in the coupling reaction, (2) must be stable under the conditions employed in rèmoving the a-amino protecting group, and (3) must be readily removable upon completion of the desired amino acid sequence under reaction conditions ~hat will not alter the structure of the peptide chain.

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~-585~ -13-It will be apparent to those skilled in the art that the protecting groups known to be useful for ~eptide synthesis will vary in reactivity the agents employed for their removal. For example, certain protecting groups, such as triphenylmethyl and 2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleaved under mild acid conditions. Other protect-ing groups, such as t-butyloxycarbonyl, t-amyloxycar-bonyl, adamantyloxycarbonyl, and _-methoxybenzyloxy-carbonyl, are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid, for their removal. Still other protecting groups, such as benzyloxycarbonyl, halobenzyloxycarbonyl, _-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less labile and require strong acids, such as hydrogen fluoride, hydrogen bromide, or boron tri-fluoroacetate in trifluoroacetic acid, for their removal.
Illustrative examples of amino acid protect-ing groups are set forth below.
A~ For an ~-amino group, protection may include (a) acyl-type groups, such as formyl, trifluoracetyl, phthalyl, p-toluenesulfonyl (tosyl), benzene-sulfonyl, nitrophenylsulfenyl, and the like; tb) aromatic urethane-ty~e groups, such as benzyloxycarbonyl and substi-tuted benzyloxycarbonyl, such as, for example, _-chlorobenzyloxycarbonyl, 7~

p-bromobenzyloxycarbonyl, ~-nitrobenzyl-oxycarbonyl, and ~-methoxybenzyloxycar-bonyl, o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2,6-dichloro-benzyloxycarbonyl, and the like; (c) aliphatic urethane-type groups such as t-butyloxycarbonyl, t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenylyl)-isopropyloxycarbonyl, allylo~ycarbonyl, and the like; (d) cycloalkyl urethane-type groups such as cyclopentyloxycar~
bonyl, cyclohexyloxycarbonyl, cyclo-heptyloxycarbonyl, adamantyloxycarbonyl, and the like; (e) thiourethane-type groups such as phenylthiocarbon~l; (f) alkyl-type groups such as triphenyl-methyl, and (g) trialkylsilane groups, such as trimethylsilane. A preferred a-amino protecting group is t-butyloxy-carbonyl (BOC).
B. For the -amino protecting group present in lysine, protection may be by any of the groups mentioned hereinabove for protection of an ~-amino group. Typical groups include, for example, benzyloxy-carbonyl, p-~chlorobenzyloxycarbonyl, ~-bromobenzyloxycarbonyl, o-chloro-benzyloxycarbonyl, 2,6-dichlorobenzyl-oxycarbonyl, 2,4-dichlorobenzyloxy-carbonyl, o-bromobenzyloxycarbonyl, p-~7~0~

nitrobenzyloxycarbonyl t-butyloxycar-bonyl, isopropyloxycarbonyl, t-amyl-oxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyl-oxycarbonyl, adamantyloxycarbonyl, p-toluenesulfonyl, and the like. The preferred E-aminO protecting group i5 _-chlorobenzyloxycarbonyl (ClBzl).
C. For the imidazole group of histidine, protection may be by the groups mentioned above for an a-amino group, but pref-erably are benæyl, _-toluenesul~onyl (tosyl), 2,~-dinitrophenyl and benzyl-oxymethyl.
D. For the guanido group of arginine protection may be by the groups mentioned above for an ~-amino group, but pref-erably is _-toluenesulfonyl (tosyl~.
E. For the hy~roxyl group of serine, homo-serine, threonine, or tyrosine, pro-tection may be, for example, by Cl-C4 alkyl, such as methyl, ethyl, and t-butyl; benzyl; substituted benzyl, such as _-methoxybenzyl, _-nitrobenzyl, p-chlorobenzyl, and o-chlorobenzyl;
Cl-C3 alkanoyl, such as formyl, acetyl, and propionyl; triphenylmethyl; or benzoyl. The preferred hydroxyl pro-tecting group is benzyl (Bzl).

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F. For the carboxyl group of aspartic acid or glutamic acid, protection may be, for example, by esterification using groups such as benzyl, t-butyl, cyclohexyl, cycl-opentyl, and the like. The current groups of choice are cyclohexyl and cyclopentyl.
Upon completion of the desired peptide sequence, the protected peptide must be cleaved from the resin support, and all prot~cting groups must be removed. Thus, the process concerns the deblocking of a correspondingly protected peptide of formula I with a deblocking agent.
An example of a correspondingly protected peptide is one of formula II
(R5)(R6)Thr-a-(R6)b-(R8)Lys-(R6)d-(R7)e-f-(~g)g-(R )h-(R8)i-(Rlo)k (Rll)l-(Rlo)m (R8) 7 6 u-Pro-(R6)v-(R9)w-(Rlo)x (Rlo)Y E (R6) Z] r Z

R5 is hydrogen, Cl-C3 alkyl or an a-amino bLocking groupi R6 is hydrogen or an hydroxy protecting group;
R7 is hydrogen, an amino protecting group, or an hydro~y protecting group;
R8 is hydrogen or an amino protecting group;
Rg is hydrogen, an amino protecting group, a ~!ydroxy protecting group or a carboxyl protecting group;
Rlo is hydrogen or a carboxyl protecting group;

~ . .. ... ...
,, , ", .. ~ . .... , .,i . . .. . , ~ , . ... .

1~971~

Rll is hydrogen, a carboxyl protecting group or an amino protecting group;
Z is hydroxy, NR3R4 in which R3 and R4 independently are hydrogen or Cl-C3 alkyl, or a resin support;
subject to the limitation that when Z is hydroxy or NR3R4, then at least one of R5 to Rll must be other than hydrogen.
The cleavage reaction and removal of the protecting groups may be accomplished simultaneously or stepwise. When the resin support is a chloromethylated polystyrene resin, the bond anchoring the peptide to the resin is an ester lin~age formed between the free carboxyl group of the C-terminal moiety and one of the many chloromethyl groups present on the resin ma-trix.
It will be recognized that the anchoring bond can be cleaved by reagents which are known to be capable of breaking an ester linkage and of penetrating the resin matrix. One especially convenient method is by treat-ment with a strong acid, such as HCl in glacial aceticacid, formic acid (95% or more), or liquid hydrogen fluoride, preferably liquid hydrogen fluoride. The strong acid not only will cleave the peptide from the resin but will also remove all protecting groups. Hence, use of this reagent will directly afford the fully deprotected peptide. ~hen it is desired to cleave the peptide without removing protecting groups, ~the pro-tected peptide~resin can undergo methanolysis to give the pro~ected peptide in which the C-terminal carboxyl group is methylated. The methyl ester can then be ~97P.~)~
X 5~5~ -18-hydrolyzed under mild, alkaline conditions to give thefree C-terminal carboxyl. The protecting groups on the peptide chain then can be removed by treatment with a strong acid, such as liquid hydrogen fluoride. A
particularly useful technique for methanolysis is that of G. Moore et al., Peptides, Proc. 5th Amer. Pept.
Symp., M. Goodman and J. Meinhofer, Eds. r John Wiley, N.Y., 1977, pp. 518-521, in which the protected peptide-resin is treated with methanol and potassium cyanide in the presence of crown ether.
Another method for cleaving the protected peptide from the resin is by ammonolysis or b~ treat-ment with hydrazine. The resulting C-terminal amide or hydra~ide can be hydrolyzed to the free C-termi`nal carbo~yl r and the protectiny groups can be removed conventionally.
It will also be recognized that the protect-ing group present on the N-terminal a-amino group may be removed preferentially either hefore ox after the protected peptide is cleaved from the re~in support.
For the sake of convenience and understand-ing, the amino acids referxed herein ars described both by their approved shorthand three-letter and single-letter desi~nations.
These designations are as ollows:

3-letter l-~etter Alanine Ala A
Arginine ~rg R
Asparagine Asn ~7~

3-letter l-letter Aspartic Acid Asp D
Glutamic Acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Homoserine Hs2 Isoleucine Ile Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
lS Threonine Thr T
Tyrosine Tyr Y
Valine Val V
Using the convenient single-letter amino acid designation, the following peptides are illustrative of the compounds or formula I. ~lthough not desigrlated in the compounds illustrated below, it will be recogni2e~
that any of the depicted compounds may co:Mpxise any o~
the folLo~-ing:
tl) an alkyl group at its amino terminal nitrogen;
(2~ an alkyl substituted or unsubstitutPd ami.de at its carboxyl terminal; and (3) an added homoserine at its carbo~yl terminal, which homoserine may be derivatized by dehydra-3~ tion to its corresponding iactone.

7P.q;l ~3 T-I-S-K-A-K-G-Q-P-R-E-P-Q-V-Y-T-L-P-P-S-R-E-E;
T- I -S -K-A-K-G-Q-P-R-E-P-Q-V~Y-T-L-P-P-S-R-D-E;
T-I-S-K-T-K-G-Q-P-R-E-P-Q-V-Y-T-L-P-P-S-R-E-E, T-I-S-K-T-K-G-Q-P-R-E-P-E-V-Y-T-L-P-P-S-R-E-E;
T-I-S-K-A-K-G-Q-P-R-E-P-Q-V-Y-T-L-P-P-S-Q-E-E;
T-I-S-K-A--K-G-P--P-R--I-P-Q-V-Y-L.-L-P--P-P-R-D-E;
T-I-S-K-T-K-G-Q-P-R-F.-P-Q-V-Y-T-L-P-P-S-R-E-G;
T-I-A-K-V-T--V-N-T-F-P-P-Q-V-H-L-L-P-P-P-S-E-E;
T-L-S-K-S-G-N-T-F-R-P-Q-V-H-L-L-P-P-P-S-Q-E-L;
T-I-S-X-S-R-G-Q-P-R-E-P-Q-V-Y-T-L-G-P-S-R-D-Q;
T-L-A-K-V-G-V-S-F-L-M-P-E-II-Y-L-I-P-P-P-S-D-L;
T-I-A-R-P-G-V-K-P-R-E-K-E-V-Y-T-L-P-P-S-R-E-E;
~5 T-I-S-Y-T-R-G-Q`-P-R-E-P-Q`-V-Y-T-L-P-P-S-S N-L;
T-L-S-K-~J-K-G-Q-P-R-E-P-Q-V-Y-T-M-P-P-S-R-E-E, T- I-S~K-P-G-G-Q-P-R-P-P-E-V-Y-T-L-P-P-3Q S-R-E-F.;

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T-I-S-X-S T-G-T-P-R-I-P-V-V-Y-T-L-P-P-S-Q-E-E;
T-I-S-K-A-K-V-S-P-R-A-P-Q-V-H-L-L-P-P-P-R-D-E;
T-I-S-K-T-T-G-Q-P-R-A-P-V-V-Y-T-L-P-P-S-R-E-G;

T-I-A-K-V-T-V-T-T-F-P-P-Q-V-H-L~-G-P-P-S-~-Ei T-L-A-K-T-G-N-T-F-R-P-Q-V-H-H-L-P-P-P-1.0 S-Q-E-L;
T-I-S-K-P-R-V-Q E'-R-M-P-V-V-Y-T-L-G-P-S-R-D-Q;
T-L-A-K-V-G-V-S-F-L-E-P-E-H-Y-L-I-P-P-P-R-E-E; and the like.
The compounds of formula I, for use in modu-lating the i~une response of warm-blooded animals, have wide applicability. They can be used, for example, in treating cancer, parasitic infections, and a wide range of autoimmune diseases.
The compounds of formula I can be used in a variety of pharmaceutical cornpositions and forrnulat.ions compri3ing aS act.ive ingredient a compo~n~ of ~ormula I
with one or more physiologically-acceptable carriers, and can ~a administered by a variety ~ convelltional routes, such as lntramuscular, intravenous, subcu-taneous, intraperitoneal, and oral.
In ~?~ministering the compounds of formula I
pare.nterally or intraperitonealiy, the pharmaceutical ~orms sultable for injection include sterile aqueous solutions or dispersions a~d sterile powders for re-. .

1~7P,~

constitution into sterile injectible solutions or dispersions. The carrier can be a solvent or dispers-ing medium cont~iningr for example, water, ethanol, polyol (for example glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereoL, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of sur-lo ~actants. Prevention of'the action o microorganismscan be ensured by various antibacterial and antifunga agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodi~n chloride, and the like. Prolonged absorption of the in~ectable pharmaceutical form can be brought about by the use of agents delaying absorption, ~or example, aluminum monostearate and gelatin.
Sterile injectable'solutions can be prepared by incorporating the compounds of ormu~a I in the required amount of the appropriate solvent with various of the other ingredients, as desired.
I desired, and for more ef~ctiv2 distribu~-tion, the compounds of formula I can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
As noted, the compounds o~ ~ormula I can also be administered orally. They can be used with an inert d luent or a pharmaceutical carrier which can take ~he form of capsules, tabieLs, suspensions, , 11~7,P.~

X-585~ -23-emulsions, solutions, dispersible powders, and the like. Moreover, the compouncs of formuia r can be a~min;stered either alone or as a mixture of a plural-ity of active ingredients.
Doses of the compounds of formula I are administered to the recipient for a period during which molulation of the i~nune response is desired. The weight of the recipient and mode of administration will have an influence upon the size of the dose necessary 1~ to induce a particular response. Generally, for parenteral or intraperitoneal administration, the dose will be from about 200 llg. to about 10 mg. per kilogram (kg.) body weight of the recipient per da~ throu~hout the period of desired i~nune response modulation.
lS P~eferably, the compound is administered in an amount from about 500 ~g~ to a~out 5 mg. per kg~ body weight daily .
It is especially advantage~us to formulate the compounds of formula I in unit dosage form for ease of admini5tration and uniformity o dosa~e. Unit dosage Eorm as used herein refers to a physically discrete unit suited as unitary dosages fo~ the sub~ect t:o be tr:eated. Each unit contains a predetermined quarltity oE the compound calculated to produce the desired therapeutic efect in association with the pharmaceutically acceptable carrier. The specific unit dosage form is dictated b~ and dire~-tly dependent upon ~a~ the unique characteristics of the particular com~osition and (b) the particular thera~eutic e~fect to be achieved.

7~30~

- The following examples are illustrative of this invention. They are not intended to be limiting upon the scope thereof.
Example l---Preparation of L-Threonyl-L-isoleucyl-L-seryl-L-lysyl-L-alanyl-L-lysyl-glycyl-L-qlutaminyl-L-prolyl-L-ar~inyl-L-glutamyl-L-prolyl-L-glutaminyl-L-valyl-L~tyrosyl-L-threonyl-L-leucyl-L-prolyl-L-prolyl-L-seryl-L-arginyl-L-glutamyl-L-glutamyl-L-homoserine lactone from IgG, and Its Biological Potency.
IgGl ~Pet), lO0 mg., obtained from Dr. Hans L. Spiegelbe~, Scripps Clinic and Research Foundation, was incubated with l.2 mg. of plasmin for 24 hours at 31'C. and pH 7.2. The resulting di~ested IgGl then was se~ara~ed on a~;ephade:; ~-lOOIcolu~n ~l.5 cm. x 75 cm.) usir.y O.OlM phos~hate buffer, pH7, as eluant.
. Two peaks were seen. Each was collected and analyzed for its ability to induce murine splenic B
cells to secrete polyclonal antibody. The method is described in E. L. Morgan and W. O. Weigle, J. I~mun.
124, 1330 ~l980). As the following shows, only Peak II
con,ains b:iolo~ically active mat~rial.
PFC/lO
Peak I 10 + 3 Peak II 98 + l ~ he material obtained from Peak II ~50 m~ A !
was dissolve~ in 0.~ ml. of 88% formic acid. ~ lO0-~01~ eXCeSS (W~W) OL CNBr was added, and the mixt~re was allowed to rsact ove~niqht a~ rcom temperature.

* Trademark 1~7~

~-5854 -25-The resulting mixture was frozen on dry ice and lyophi-lized to remove excess CNBr.
The product from lyophilization was subjected to molecular exclusion high performance liquid chroma-tography (HPLC) using two protein I-60 columns (7.8 mm.
x 30 cm) (Waters Associates, Inc., Medford, Mass.) in tandem, in line with a Waters HPLC apparatus. The mobile phase buffer consisted of O.lM NH4~iC03. Elution was carried out at a flow rate of 1 ml./minute and was monitored at 210 nm. Two peaks, designated Pools 1 and 2, were recovered and were assayed for ability to induce a polyclonal antibody response. The following results were obtained:

Pool 1 11 + 1 Pool 2 151 + 15 From the above, it is evident that the active frasment is in Pool 2.
The material from Pool ~ WâS subjected to re~erse phase HPLC usin~ a ~I,S-Zorb~ (Dupont Instru-ment, Wilmin~on, Del.) column (4.6 mm. x 25 cm.).
Peptide elution was achieved using a linear gradient of 0-100% buffer "B" in buffer "A" developed 45 minutes at a flow rate of 1 ml./min. Buffer "A" was composed of 5% CH3C~l in 0.1% H3PO4 and buffer "B" of 95% C~3CN in 0.1% H3PO~. Tne elution was monitored by absorbance at ~1~ nm.

* Trademark :

7~

X-5854 ~26-Five pools (A-E) were collected, and each was assessed for its ability to induce murine splenic B
celLs to secrete polyclonal antibody. Following are the re~ults which were ohtained:

Pool A 95 + 2 Pool B 19 * 6 Pool C 10 ~ 4 Pool D 3 + 3 Pool E 2 + 1 The above results reveal that only the mat~-rial from Pool ~ had the capacity to induce polyclonal antibody production.
The amino acid composition of the peptide of Pool A was determined, using a Beckman Model 121M amino acid analy2er~ The sample was hydrolyzed for 24 hours in an evacuated ampoule 0.5 ml. lN HCl. 1 ml. 88~
phenol, and 1 ml. 2-merc2ptoet~anol. The follvwing results, on a mole/mole basis, were obtained:
~ r, 2.02; S~r, 1.98; Glu, 5.01; Pro, 4.41;
Gly~ 1.08; Ala, 1.01; Val, 0.97; Ile, 0.98; Leu, 1.07;
Ty., 0.57; Lys, 1.97; Arg, 2.04; His, 0.11; .~sp, 0.0;
Phe, 0.0; Me~ 0.0*

*Methionine was converted to homoserine lac-tone by CNBr cleavage and therefore was not quantitatedA

3~

7t~

Example 2--Preparation of L-Threonyl-h-isoleucyl~L-seryl-L-lysyl-L-alanyl-L-lysyl-L-glycyl-L-glutaminyl-L-prolyl~L-arginyl-L-glutamyl-L-prolyl-L-glutaminyl-L-valyl-L-tyrosyl-L-threonyl-L-leucyl-L-prolyl-L-prolyl-L-seryl-L-arginyl-L glutamyl-L-glutamic acid.
A. N-t-Butyloxycarbonyl-L-(~-cyclopentyl)glutamic acid hydro~ymethyl-polystyrene resin ester To 50 ml. of 95~ ethanol were added 3.784 g.
(12.0 mmol.) of N-t-butyloxycarbonyl-L-(y-cyclopentyl)-glutamic acid. To the resulting solution then were added 12.0 ml. of lN cesium bicarbonate solution with accompanying C02 evolution. The mixture was evaporated in vacuo to an oil. The oil was dissolved in a mixture 15 of ben~ene and 95% ethanol, and the resulting solution c was evaporated again. This water-azeotroping procedure was repeated until the water had been removed with formation of a white solid.
The cesium salt of the protected glutamic 20 c~cid was dissolved in 200 ml. of N,N-dimethyl~ormamide (D~F), and ~ne mixture was placecl in a vessel equipped with a stirrer aIld a àryin~ tube and in the presence of a dry N2 atmQspheL-e. With maintenance of tlle N~
atmosphere, 20.0 g. of chloromethylated polyst~r~ne 25 resin (.~lerri~ield resin) (about 0.7 mmol. chlorine per gram resin) were added. The ~ixture was stirred at 50C. for about 72 hours.
The resin was collected by filtration, washed three ~imes with a sequence or 85~ DMF:~S~ ~120 follo~led 3Q by DMF, ~hree times with 95~ ethanol, and once witn :~L9~

X-5~54 -28-DMF. The resin then was dried in vacuo to obtain about 21 grams which, by analysis, showed about 0.2 mmol.
Glu/g. resin.
The resin was suspended in 200 ml. of DMF, and 5.96 g. (31 mmol.) of cesium acetate ~hydrate) were added. The mixture was stirred in a dry N2 atmosphere at 60C. for about 7Z hours.
The resin was collected by filtration and washed with DMF, 85% DMF, and 95~ ethanol using a sequence similar to that previously described. The fines were removed by suspending the resin in C~C13 (6 portions) in a separatory funnel and drawing off the CHCl3. The resin was filtered, washed wit~ 95g ethanol, and dried ov~rnight in vacuo at 45C.
__ ___ B. N-t-ButyLoxycarbonyl-L-(O-benzyl)threonyl-L-iso-leucyl-L-~O-benzyl)seryl-L-(N~-o-chloroben~yloxy-carbonyl)lysyl-L-alanyl-L-~N~-o-chlorobenzyloxy-carbonyl)lysyl-glycyl-L-glutaminyl-L-prolyl-L-(N-_-tosyl)arginyl-L-(y-cyclopentyl)glutamyl-L-prolyl-L-glut~minyl-L-v21yl-L [0-(2,6-~ichlorobe.nzyl)]-tyrosyl-L-(O-~enzyl)threonyl-L-leucyl-L-prolyl-L-prolyl-L-(O-benzyl)seryl-L-(N-p-tosyl)argi.nyl-L-(y-cyclopentyl~.glutamyl-L-(y-cyclopentyl)-glutamic acld hydro~ymethyl-polystyrene resin es~er N-t-Butyloxycarbonyl-L.-(y-cyclopentyl)-glutamic acid hydroxymethyl-pGlystyrene resin ester ., 1197P~Q

(20.15 g.), as prepared in Part A, was placed into aBeckman 990B peptide synthesizer and treated with the following sequence of protected amino acids using one or more of the schedules set forth below.
1. N-t-butyloxycarbonyl-L-(y-cyclopentyl)-glutamic acid Schedule A - 5.05 g.
Schedule B - 3.78 g.
2. N-t-butyloxycarbonyl-I.-(N-p-tosyl)argirline Schedule A - 6.85 g.a Schedule B - 5.14 g.
Schedule B - 5.14 g.b 3. N-t-butyloxycarbonyl-L-(0-benzyl)serine Schedule A - 4.72 g.
Schedule B - 4.72 g.

4. N-t-butyloxycarbonyl-L-proline Schedule A - 3.46 g.
_ Schedu]e B - 3.46 g.

5. N-t-butyloxycarbonyl-L-leucyl-L proline Schedule A - 5.27 g.
Schedule B - 5.27 g.

6. N-t-butyloxycarbony:L-L-~O-benzyL)threonine Schedule A - 4.95 g.
Schedule B - 4.95 g.
~

~7~

7. N-t-butyloxycarbonyl-L-[0-(2,6-dichloro-benzyl)]tyrosine Schedule A 7.04 g.
Schedule B - 7.04 g.

8. N-t-butyloxycarbonyl-L-valine Schedule A - 3.48 g.
Schedule B - 2.60 g.
Schedule 3 - 2.60 g.b

9. N-t-butyloxycarbonyL-L-glutamine, p-nitrophenyl ester Schedule C - 5.88 g.
Schedule D - 5.88 g.

10. N-t-butyloxycarbonyl-L-proline Schedule A - 3.46 g.
Schedule B - 3.46 g~

11. N-_-butyloxycarbonyl-L-(y-cyclopentyl)-0 glutamic acidSchedule A - 5~5 g.
schedule B - 3.78 g.

12. N-t-bu-tyloxycarhcnyl-L-(N-p-tosyl.)~
arglnlne Schedule A - 6.85 y.a Schedule B - 5.14 g.
Schedule B - 5.14 g.b

13. N-t-butyloxycarbonyl-L-proline ~ Schedu1~ A - 3~46 g.
Schedule B - ~.46 g.

~-5854 -31-

14. N-t-butyloxycarbonyl-L-glutamine, p-nitrophenyl ester Schedule C - 5.88 g.
Schedule D - 5.88 g.

15. N-t~butyloxycarbonyl-glycine Schedule A - 2.80 g.

16. N-_-butyloxycarbonyl-L-(N --o-chloro~
benzyloxycarbonyl)lysine Schedule A - 6.64 g.
Schedule B - 6.64 g.

17. N-t-butyloxycarbonyl-L-alanine , Schedule .~ - 3.03 g.
lS 18. N-t~butyloxycarbonyl-B-tNE-o-chloro-benzyloxycarbonyl~lysine Schedule A - 6.64 g.
Schedule B - 6.64 S~
19. N-t-butyloxycarbonyl-L-(~-ben~yl)~serine Schedule A - 4~72 g.
Schedule B - 4.72 g.
20. N-t-butyloxycarbonyl-L-isoleucine Schedule A - 3.70 g.
Schedule B-- 3.70 g.

7P~
~~5854 -32-21. N-t-butyloxycarbonyl-L-(O-benzyl)threonine Schedule A - 4.95 g.
- Schedule B - 4.95 g.
Footnotes:
aj 1:9 DMF:CH2C12 b) DMF
SCHEDULE A
1~ 1. Wash three times for three ,ninutes each with 6.5 ml. of CH2C12 per gram resin.
2. To remove the t-butyioxycarbonyl a-amino protecting group, treat twice for twenty minutes each with 8.4 ml. per gram resin of a mixture oE tri-fluoroacetic acid (29~, CH2C12 (65%~, and triethyl-silane (6~).
3. Wash three times for three minutes each wlth 6.5 ml. of CH2C12 per gram resin.
4. Wash three times for three minutes each ~0 with 6.5 ml. per gram resin of a mi.cture of 90%
_-BuOH and 10~ t-~mOH.
5. ~ash three times for three minutes each with 6.5 ml. of CH2C12 per gram resin.
6. For neutrali~ation, treat thre~ tim~s for three minutes each with 6.5 ml. per gram resin o~
a mixture of 4~ diisopropylethylamine in CH2C12.
7. Wash three ti~.es for three minutes each with 5.5 ml. of CH~C12 per gram resin.
8. Wash three times for 1:hree minutes each wi h 6.5 ml. per gxam resin of a mix~ure of 9Q~
t-auoH and lQ% t-AmOH.

1~!37P~

X-5854 ~33~

9. Wash three times for three minutes each with 6.5 ml. of C~2C12 per gram resin.
10. To couple the amino acid, treat with the protected amino acid and N,N'-dicyclohexylcarbo-diimide (50~ on molar basis relative to the aminoacid) in 80 ml. of CH2C12 for 110 minutes.
11. Wash three times for three minutes each with 6.5 ml. of CH2C1~ per ~ram resin~
12. Wash th ee times for three minutes each with 6.5 ml. p~r gram resin of a mixture of 90% t-8uOEI
and 10% t-AmOH.
13. Wash three times for three minutes each with 6.5 ml. of CH2C12 per gram resin.
14. For neutralization, treat three times for three minutes each with 6.5 ml. per gram resin of a mixture of 4~ diisopropylethylamine in CH2C12.
15. Wash three times for three minutes each with 6.5 ml. of CH2C12 per qram resin.
16. Wash three time~ ~or three minutes each ~; 2~ with 6.5 ml. per gram resin of a mi~ture o ~0~ t-~uOH
and 10% t-AmOH.
17. Wash three timas for three minutes each with 6.5 ml. of CH2C12 per gram resin.
SCHEDULE B
2~
1. Wash three times for three minutes each with 6.; ml. of DMF per gram resin.
2. To couple the amino acid, treat with the protected amino acid and N,N'-dicyclohexylcarbodiimide 3n (50~ on molar basi3 relative to the amino acid~ in 80 ml. of 1:1 nMF:C~2C12 for ilO minutes.

7~

X-5854 ~34~

3. Wash three times for three minutes each with 6.5 ml. of DMF per gram resin.
4. Wash three times or three minutes each with 6.5 ml. of CH2C12 per gram resin.
5. Wash thr~e times for three minutes each with 6.5 ml. per gram resin of a mixture of 90~ t-BuOH
and 10% t-AmOH~
6. Wash three times for three minutes each with 6.5 ml of CH2Cl~ per gram resin.
7. For neut:ralization, treat three times for three minutes each with 6.5 ml. per gxam resin of a mixture of 4~ diisopropyletllylamine in CH2C12.
8. Wash three times or three minutes each with 6.5 ml. of CH2C12 per g~am resin.
9. Wash three times for three minutes each with 6.5 ml. per c3ram re~in of a mixture of 90% t-BuO~
and 10% ~-AmOH.
10~ Wash three times for thre~ minutes each with 6.5 ml. of C~2C12 per gram resin.
SCHEDULE C
Schedule C is the same as Schedule A in all .espects e~cept as to the amino acid coupling Step 10.
Step 10 of Schedule A is replaced b~ a sequence of thr~e steps as ollows:
~5 a) Wash thxee times ~or three minutes each with 6.5 ml. of DMF per gram resin~
~ b, To couple the amino acid, treat with the protected ami~o acid, active ester, in 80 ml. of a 3:5 mixture of DMF and CH~Cl~ for 10 h~urs.
c) Wash thrae times for three minuies each with 6.5 ml. of DMF per gram Lesin.

7~ 0 X-5854 ~35-SCHEDULE D
Schedule D is the samP as Schedule ~ in all respects except as to the amino acid coupling Step 2.
Step 2 of Schedule D is as follows:
To couple the amino acid, treat with the protected amino acid, active ester, in 80 ml. of a 5:3 mixture oE DMF and CH2C12 or 10 hours.
C. L-Threonyl-L-isoleucyl-L-seryl-I,-lysyl-I,-aLanyl-L-lysyl-glycyl-L-glutaminyL-L-prolyl-L-arginyl-L-glutamyl-L-prolyl-L-glutaminyl-L-valyl-L-tyrosyl-L-threonyl-L-leucyl-L-prolyl-I.-prol~l-L-seryl-L-arginyl-L--glutamyl-L--glutamic acid To 16.5 g. of the resin product of Par~ B
1~ were adde.d 23 ml. of anisole and 23 ml. of ethyl mercaptan. The mixture was frozen in a liquid N~ bath, and 250 ml. of liquid HF were transferred into t~e mixture by distillatior.. The liquid N2 bath was replaced by an ice water bath, and the mixture was stir~ed ror 2 hours at 0C. The HF then was removed in v cuo, and ethyl e~her was added to the remainder to pxecipitate the peptide product. The precipitate was filtered, washed with ether, and air dried. Th0 precipitate, comprisin~ deblocked pepticle and resin, was slurr~.ed in 2S ml. of 50~ acet.ic acid and fi].tered.
The residue (resin) then w~s rinsed with 10 ml. of 505 acetic acid a~ter which it was reslurried in 0.2 M
acetic aci.d, filtered and rinsed with 10 ml. oE 50~
acetic acid. The resin then was extracted four times with 30 ml. porLions of 0.2 ;~ acetic ~cid and washed ~7~00 with water. The washings and extracts were combined and filtered through glass fiber to provide a total of 350 ml.
of 6.9~ aqueous acetic acid containing the deblocked peptide product.
The acetic acid solution containing deblocked peptide product was applied to a 10 x 225 cm ISephadex G-25F' column which had been equilibrated with degassed 0.'2M acetic acid. The column was eluted with 0.2M acetic acid by gravity flow for 16 hours and at 773.3 g./cm.2 for the xemai~der. The optical density (OD~ if`the eluant was monitored with an 'Isco' Model UA-5 using a 1.0 cm. flow cell and a filter for monitoring 280 nm. The following pools, described in terms of their elution and total volumes, were prepared:
'Pool'No. E'lut'ion Volume, ml. Pool Volume, ml.

III 10420-10~70 550 Pool II, containing about S00 mg., was subjected to preparative reverse phase high performance liquid chromatogrpahy (HPLC) under the following conditions:

Column: 5.0 cm. x 60 cm.
Packing: C18 LP-l~ capped Solvent: - 15~ CH3CN, 0.05 M
HCOONH4, to pH 4.2S with HCOOH
Flow Rate: about 10 ml./ min.
Monitored at 280 nm * Trademark ** Trademark , . ., . , . , ~ . . . . . . .. . .

7P~O~
X-5854 ~37~

A peak appeared at 1985 ml. to 2379 ml. elution volume. It was collected, lyophilized, and estimated by W absorption to contain about 180 mg. of peptide.
The recovered peptide, dissolved in 50 ml. of 0.01 N HCl containing 7M urea, was subjected to ISepha-dex G-50F' chromatography under the following conditions:

Column: 5.0 cm. x 215 cm.
Packing: ISephadex G-50' Fine Solvent: lM acetic acid Flow Rate: about 2.5 ml./min.

The product was collected at 2690 ml. to 3120 ml.
elution volume, lyophilized, and estimated by UV absorption to contain about 80 mg. o peptide.
Amino acid analysis: Thr, 1.93; Ser, 2.03; Glu, 5.28; Pro, 4.19; Gly, 0.99; Ala, 1.00; Val, 0.99; Ile, 0.93; Leu, 1.02; Tyr, 0.98; Lys, 2.00; Arg, 2.04.
The above results represent the average of 21 20 hour and 72 hour hydrolysates ratioed to 0.25 of (Gly Ala & Lys), with the exception that Thr and Ser were extrapolated to zero hours hydrolysis and Val was extrapolated to infinite hydrolysis time.
The ability o compunds of formula I to modulate an immune response is demonstrated using a range o assays.
In each of the following assays, the compound prepared by Example 2 is used.

* Trademark 7P~

A. Polyclonal Antibody Response Assay This assay measures the activity of test compound in potentiating an in ~itro polyclonal response using mouse spleen and human peripheral blood cells.
1. Mouse--For the generation of the polyclonal plaque-forming cell (PFC) response, mouse spleen cells were suspended to a concentration of 6 x 106/ml. in RPMI
1640 ~Flow Laboratories, Rockville, Md.) supplemented with 2mM L-~lutamine, 1% BME vitamins (Grand Island Biological Co., N.Y~), 100 u~its penicillin, lOO ~g.
streptomycin, 5 x 10 5M 2-mercaptoethanol (2-ME), 0.5~
fresh normal mouse serum, and 7.5% fetal calf serum IFCS).
A predetermined quantity of test compound was added Duplicate cultures of 6 x 105 cells/0.3 ml. were incubated in microtiter plates at 37C. in 5~ CO2. The duplicate cultures were harvested on day 3 and assayed for a response to 2,4,6-trinitrophenyl (TNP) by the slide modification of the plaque assay described in N.K. Jerne and A.A. Nordin, Science 140, 405 (1963). Heavily conjugated TNP-sheep red blood cells (TNP-SRBC) were prepared according to the method described by J. Kettman and R.W. Dutton, J. Immunol.
104, 1558 (1970) and were used as the indicator red blood cell (RBC). Guinea pig serum was the source of complement (C) to develop the direct or IgM plaques. Results of the pla~ue-forming assay are shown in the ~ollowing Table I and are expre~sed as mean PFC/106 original cells of duplicate pools + standard error.

* Trademark 1~7P~

Table I

Pol'yclona'l Antibody Pro'ducti'on by' Mouse 'Spleen Cells Direct Anti-TNP
Compound, (~g/ml)-. PFC/.10 + SE
_ 6 + 7 .025 87 ~ 3 0.25 201 + 5 2.5 31 + 16 -2. Human--Thi.s àssay was carried out-using.
human peripheral blood lymphocytes(PBL). PBL was prepared by diluting heparinized peripheral blood with two volumes of phosphate-buffered saline (PBS), 0.001 M
phosphate pH 7.2,0.15 M NaCl. Lymphocytes were sepa-rated by 'Ficoll-Paque' (Pharmacia) gradient centrifuga-tion as described in E. L. Morgan and W. O. Weigle, ~.
Exp. Med. 154, 778 (1981).
PBL suspensions were enriched for B and T
cèlls by the neuraminidase-treated SRBC rosette tech-nique described in Morgan and Weigle, s~ . The non-rosetting cells were defined as B cell and monocyte enriched and the rosetting cells as T cell-enriched.
The T cell populations were sub~ected to 2000 R of irradiation prior to use in the assay.
The human polyclonal antibody response was measured using a mixture of 1 x 105 B cells and 2 x 105 irradiated T cells to which were added a predetermined quantity of test compound. The cells were suspended and treated as in the above-described mouse polyclonal * Trademark ~`71~

assay, with the exception that the cultures were harvested on day 6 instead of day 3. The results are provided in the following Table II.
Table II

Polyclonal' Antibody''Pr'oduction by Human'Peripheral Blood Lymphocytes Ig-Secreting Cells/106 - Compou~d(~g/ml) B Cells + SE
- 65 +
.003 1,217 + 37 B. In Vitro Anti-SRBC Adjuvant Assay 1. Mouse--Spleens were removed from mice which four to six weeks previously had been injected i.p. with 0.1 ml. of a 10% suspension of SRBC. A modified Mishell-Dutton culture system was employed for genexation of antibody producing cells. Cells were suspended to a con-centration of 6x 106/ml. of RPMI-1640 supplemented with 2 m~l L-glutamine, 1% BME vitamins, 100 U penicillinr 100 ~g.
streptomycin, 5 x 10 M 2-ME, 7.5% FCS, and 0.5% fresh normal mouse serum. The spleen cells at a concentration of 6 x 105 along with 5 x 104 SRBC and a predetermined amount of test compound were cultured in 0~3 ml. final volume in flatbottom microtiter plates for 4 days at 37C. in 5~ CO
The direct anti-SRBC response was measured by the slide modification of the Jerne and Nordin plaque assay. The results, shown in the following Table III, are recorded as direct anti-SRBC PFC/106 original cells ~ standard error.

i.`''.

7~0~

Table III

Enhancement of the Mouse in Vitro Anti-SRBC Response Direct Anti-SRBC
Compou~d(~gjml) SRBCaPFC/106 + SE
- + 129 + 9 0.03 + 560 + 18 aS x 104 SRBC/culture 2. Human--The primary anti-SRBC response was "
carried out as described by J. Misiti and T. A. Wald-mann, J. Exp. Med. 154, 1069 (1981). Peripheral blood lymphocytes (5 x 106) were cultured in 1 ml. of RPMI
1640 supplemented with 2mM L-glutamine, 1% BME vita-mins, 100 U penicillin, 100 ~g. streptomycin~ 5 x 10 5 M 2-ME, and 10% SRBC-absorbed autologous human plasma.
The desired amount of test compound plus lx 105 SRBC
were added prior to culturing. The cultures were maintained in gas box~s~ which were rocked at 7 or 3.5 cycles/minute for 11 days. Every other day the cul-tures were fed a cockta~ mixture containing autologous plasma as described by R. I. Mishell and R. W. Dutton, _. Exp. Med. 126, 423 (1967). The direct or IgM response was measured by the Jerne and Nordin pla~ue assay on day 11 of culture. The results, shown in Table IV
following, are recorded as direct anti-SRBC PFC/culture + standard error.
X

~7P~

X-5~54 -42-Table IV
Enhancement of the Human in Vitro Anti-Sr~C Response Direct Anti~SRBC
5Compound (yg/ml) SRBCaPFC/Culture +.SE
_ +43 _ 4 0.03 +292 + 16 al x 105 SRBC/culture.
C. _ Vivo Anti-SRBC Adjuvant Assay Mice, in groups of 4, Wel`e given 0.1 ml~ of a SRBC suspension intraperitoneal].y ollowed immediately by in~ravenous injection o saline or saline containing test compound. T,he spleens were assessed for PFC to SRBC five days after immunization. The results are shown in Table V ollowin~.
Table V
Enhan.ement of the Primary ~n Vivo ~nti-SRBC Response ~ire~ nti-SRBC
_RBCaCompo~lnd ~yg/m~ouse)PE~`C/10 t SE
+ _ 23 + 3 120 ~ 5 aO.l ml. of 0.1~ suspension was injected/mouse.

11~7P"~)~

X-5854 ~43~

D. Cell Mediated Lympholysis (CML) Assay This assay measures the ability of test compound to augment the T cell cytolytic response.
l. Induction of Cytotoxic (Effector) Cells.
Normal C57~L/5 T cells ~2.5 x lO ) are cul-tured for 5 days with 2.5 x lO irradiated (~dOOR) CBA/CaJ spleen cells at 37C. in 5% C02 (final volume -0.3 ml~) in flat bottom microti~er plate~. The culture ]o medium consisted of RPMI-l640 supplemented with 2~M
L-glutamine, 1% B~IE vitamins, lOO units penicillin, lOO mg. streptomycin, 5 x lO 5 M 2-mercaptoethanol (2~1~E), and lO~ fetal calf serum (FCS~.
2. Preparation o~ Target Cells.
lS Two da~s prior to assay, CBA/CaJ blast cells (target cells) are prepared by incubating 5 x lO5 CBA/CaJ spleen cells per well ~ith 20 ~g.,/ml. lipopoly-saccharide (LPS) in a manner as described above for normal T cells.
On the day of assay, blast cel' 5 are harvssted and labeled with 5lCr by incubating 2 x 106 ~la~ts with 200 ~Ci 5lCr (sodium chromate) for 60 ~i~nutes~ The cells then are washed and resuspended in RPMI-1~40 containl~g lO~ FCS.
3. Assay Varying numbers of effector calls are cul-tured with a constant numbex of ~,abeled target cells for 4 hours in round ~ottom 12 ~n. x 75 mm. plastic tubes. ~t tha ~nd of 4 hours, the cells are ce-1tri-3~ u~ed a~ lOOO rpm or 5 mlnutes, and th~ resultin~

. .

1~78QI~

~-5854 -4~-supernatant i~ analyzed for released 51Cr. The data, recorded as % Specific Release (SR), are de~ermined as follows:
% SR = Experimental Release - Spontaneous Release x 100 Maximal Release Spontaneous Release in which Spontaneous Release i~ the amount of 51Cr released in the absence o effector cells, and Maximal Reiease is the amount of 51Cr released after free~ing and -thawin~ target cells three times.
The results are shown in Table VI following.
Table VI
Enhancemant of CML Response Compound Ratio, Efector- %51Cr (~g./ml.) Target Cells Release - 10 12 + 2 ~.17 10 72 ~ 2 E. Enhancement o~ the Peripheral Blood Lymphocytas (PBL) Proliferati~e Response to Tetanus Toxoid .Antigen PBL (4 x 10 cells) are. cuitured with 1 ~g./ml.
tetanus toxoid in RPMI~ 40 ~upplemented with 2mM
L-cflutamine, 1~ RME vitamins, (00 units penicillin, 100 m~. streptomycin, and 10~ h~lan AB serum. The cultures were pulsed with-l ~Ci ~3}I]thymidine (TdR) on day 6, harvested ~ hours later and counted using a ~eta counter. The xesults are shown in Table;VII
~ollowin~.
3n :

97P~

Table VII
Enhancement of P~L-Induced Proliferative Response to Tetanus Toxoid Compound Tetanus [3H]TdR Uptake, (~g./ml.) Toxoid (llg.~ml.) cpm ~ SE
- ~ - 5,133 ~ 2,069 - 1 41,770 ~ 372 0.013 1 101,730 ~ 320 ~0 2~

3t)

Claims (16)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of formula I having the structure:
R-Thr-a-b-Lys-d-e-f-g-h-j-k-l-m-n-p-q-t-u-Pro-v-W-X-Y-(z)r-R1 in which each amino acid residue has an L-configuration;
R is hydrogen or C1-C3 alkyl;
R1 is OH or NR3R4 in which R3zand R4 in-dependently are hydrogen or C1-C3 alkyl;
a is Ile or Leu;
b is Ser or Ala;
d is Ala, Thr, Pro, Val, or Ser;
e is Lys, Arg, Thr, or Gly;
f is Gly, Val, or Asn;
g is Gln, Lys, Ser, Glu, Pro, Ala, Asn, or Thr;
h is Pro, Val, Thr, or Phe;
j is Arg, Leu, Pro, or Phe;
k is Glu, Ala, Ile, Met; or Pro;
l is Pro, Lys, or Gln;
m is Gln, Glu, or Val;
n is Val or His;
p is Tyr, His, or Leu;
q is Thr, Val, or Leu;
t is Leu, Ile, Met, or Pro;
u is Pro or Gly;
v is Ser or Pro;
w is Arg, Gln, Glu, or Ser;

x is Glu, Asp, Gln, or Asn;
y is Glu, Gln, Gly, or Leu;
z is Hse; and r is 0 or 1, which comprises deblocking a correspondingly protected peptide of formula I with a deblocking agent.

2. A process of claim 1 wherein the cor-respondingly protected peptide is of the formula II
(R5)(R6)Thr-a-(R6)b-(R8)Lys-(R6)d-(R7)e-f-(R9)g-(R6)h-(R8)j-(R10)k-(R11)l-(R10)m-(R8)n-(R7)p-(R6)q-t-u-Pro-(R6)v-(R9)w-(R10)x-(R10)y-[(R6)z]r-Z

R5 is hydrogen, C1-C3 alkyl or an .alpha.-amino blocking group;
R6 is hydrogen or an hydroxy protecting group;
R7 is hydrogen, an amino protecting group, or an hydroxy protecting group;
R8 is hydrogen or an amino protecting group;
R9 is hydrogen, an amino protecting group, a hydroxy protecting group or a carboxyl protecting group;
R10 is hydrogen or a carboxyl protecting group;
R11 is hydrogen, a carboxyl protecting group or an amino protecting group;
Z is hydroxy, NR3R4 in which R3 and R4 independently are hydrogen or C1-C3 alkyl, or a resin support;

subject to the limitation that when Z is hydroxy or NR3R4, then at least one of R5 to R11 must be other than hydrogen.

3. A process of claim 1 wherein the de-blocking agent is a strong acid.

4. A process of claim 3 wherein the acid is liquid HF.

5. A process of claim 3 wherein the strong acid is HCl in glacial acetic acid.

6. The process of claim 3 wherein the strong acid is formic acid (95% or more).

7. A process of claim 1 wherein when Z is a resin support the deblocking agent is methanol, followed by a strong acid.

8. A process of claim 7 wherein the de-blocking agent is methanol and potassium cyanide in the presence of crown ether, followed by a strong acid.

9. A process of claim 1 wherein the de-blocking agent when Z is a resin support is hydrazine or an ammonalysis reagent, followed by hydrolysis and strong acid.

10. A process according to claim 1 in which r is 0.

11. A compound of formula I having the structure:
R-Thr-a-b-Lys-d-e-f-g-h-j-k-l-m-n-p-q-t-u-Pro-v-w-x-y-(z)r-R1 in which each amino acid residue has an L-configuration;

R is hydrogen or C1-C3 alkyl;
R1 is OH or NR3R4 in which R3 and R4 in-dependently are hydrogen or C1-C3 alkyl;
a is Ile or Leu;
b is Ser or Ala;
d is Ala, Thr, Pro, Val, or Ser;
e is Lys, Arg, Thr, or Gly;
f is Gly, Val, or Asn;
g is Gln, Lys, Ser, Glu, Pro, Ala, Asn, or Thr;
h is Pro, Val, Thr, or Phe;
j is Arg, Leu, Pro, or Phe;
k is Glu, Ala, Ile, Met, or Pro;
l is Pro, Lys, or Glu;
m is Gln, Glu, or Val;
n is Val or His;
p is Tyr, His, or Leu;
q is Thr, Val, or Leu;
t is Leu, Ile, Met, or Pro;
u is Pro or Gly;
v is Ser or Pro;
w is Arg, Gln, Glu, or Ser;
x is Glu, Asp, Gln, or Asn, y is Glu, Gln, Gly, or Leu;
z is Hse; and r is 0 or 1, whenever prepared by the process of claim 1 or an obvious chemical, enzymatic or recom-binant equivalent thereof.

12. Compound of claim 11 wherein r is 0, whenever prepared by the process of claim 10 or by an obvious chemical, enzymatic or recombinant equivalent thereof.

13. The process for preparing H-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Glu-Glu-OH which comprises reacting N-t-butyloxycarbonyl-(O-benzyl)Thr-Ile-(O-benzyl)Ser-(N.epsilon.-o-chlorobenzyloxycarbonyl)Lys-Ala-(N.epsilon.-o-chlorobenzyloxycarbonyl)Lys-Gly-Gln-Pro-(N-p-tosyl)Arg-(.gamma.-cyclopentyl)Glu-Pro-Gln-val-[0-(2,6-di-chlorobenzyl)]Tyr-(O-benzyl)Thr-Leu-Pro-Pro-(O-benzyl)-Ser-(N-p-tosyl)Arg-(.gamma.-cyclopentyl)Glu-(.gamma.-cyclopentyl) Glu acid hydroxymethyl-polystyrene resin ester with liquid HF.

14. Compound of claim 11, which compound has the formula H-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Arg-Glu-Glu-OH, whenever prepared by the process of claim 13 or an obvious chemical, enzymatic or recombinant equivalent thereof.

15. A process according to claim 1 wherein r is 1, defined by a 23 amino acid sequence represented by residues 335-357 of the IgG molecule to which is bonded, by amide formation, at the carboxyl terminal, homoserine or the lactone produced by dehydration of homoserine.

16. A compound of the formula I as defined in claim 11 wherein n is 1, defined by a 23 amino acid sequence represented by residues 335-357 of an IgG molecule to which is bonded, by amide formation, at the carboxyl terminal, homoserine or the lactone produced by dehydration of homoserine, whenever pre-pared by the process of claim is or an obvious chem-ical, enzymatic or recombinant equivalent thereof.