CA1105923A

CA1105923A - Pentapeptides and methods

Info

Publication number: CA1105923A
Application number: CA296,484A
Authority: CA
Inventors: Gideon Goldstein; David H. Schlesinger
Original assignee: Sloan Kettering Institute for Cancer Research
Current assignee: Memorial Sloan Kettering Cancer Center
Priority date: 1977-11-15
Filing date: 1978-02-08
Publication date: 1981-07-28
Also published as: DE2804567A1; JPH0137400B2; JPS5476522A; JPS6150999A; JPS6149320B2; FR2408581A1; SE7801031L; CH640217A5; FR2408581B1; BE864122A; DE2804567C2; SE446866B

Abstract

ABSTRACT OF THE DISCLOSURE
There are disclosed new polypeptide compositions having the following amino acid sequence as the active site:
TYR-ASN-ILE-GLN-LYS
This polypeptide has the capability of inducing the differentiation of both T-precursor cells as measured by the acquisition of the thymic differentiation antigens TL and THY-l (0), as well as B-precursor cells as measured by the acquisition of receptors for complement, a distinctive marker of B cells. The peptide is thus useful in thymic function and immunity areas such as in treatment for a congenital absence of thymus. The peptide is active in very low concentrations. Also provided are derivatives of the pentapeptide, novel intermediate polypeptides, methods of manufacture of the peptides, therapeutic compositions, and methods for use of the compositions.

Description

11~5923 It is well known that many polypeptides have been isolated from various organs of animals. Until about the past decade, however, very little was known about the thymus, an organ which in man comprises about 0.8% of his body weight at birth, although it has been previously hypothesized that a neuromuscular blocking substance existed in the thymus. Despite keen interest in possible functions of the thymus and early speculation and experimentation, little was known of the function of the thymus until recently. It is now realized, however, that the thymus is a compound organ with both epithelial (endocrine) and lymphoid (immunological) compounds and thus the thymus is involved in the immunity functions of the body. The thymus is known to be a compound organ consisting of an epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells originating in haemopoietic tissues, Goldstein et al, The Human Thymus, Heinemann, London, 1969. Lymphocytes are differentiated q~

5~23 within the thymus and leave as mature thymus-derived cells, called T cells, which circulate to the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation within the thymus appears to be mediated by secretions of the epithelial cells of the thymus but difficulties with bioassays had previously hindered the complete isolation and structural characterization of any hormones which may be present.
It has been known for some time that the thymus is connected with the immunity characteristics of the body and therefore great interest has been indicated in substances which have been isolated from the thymus. In this regard, there have been published in recent years a relatively large body of articles based on scientific work relating to materials which are present in bovine thymus. In fact, the Applicants have published a number of articles which relate to research in this area. Pertinent publications may be found for example in The Lancet, July 20, 1968, pps. 119-122; Triangle, Vol. 11, No. 1, pps. 7-14, 1972; Annals of the New York Academy of Sciences, Vol. 183, pps. 230-240, 1971;
and Clinical and Experimental Immunology, Vol. 4, No. 2, pps. 181-189, 1969; Nature, Vol. 247, pps. 11-14, 1974; Proceedings of the National Academy of Sciences USA, Vol. 71, pps. 1474-1478, 1974; ell, Vol. 5, pps 361-365 and 367-370, 1975; Lancet, Vol. 2, pps. 256-259, 1975; Proceedings of the National Academy of Sciences USA, Vol. 72, pps. 11-15, 1975; Biochemistry, Vol. 14, pps. 2214-2218, 1974;
Nature, Vol. 255, pps. 423-424, 1975.
In the article by Goldstein and Manganaro in Annals of the New York Academy of Sciences, Vol. 183, pps. 230-240, 1971, there are disclosures regarding the presence of a thymic polypeptide 5~23 which causes a myasthenic neuromuscular block in animals, which is analogous to the human disease of myasthenia gravis.
Further, in this article it was discovered that two distinct ! effects were caused by separate polypeptides in bovine thymus.
One of these polypeptides, named "thymotoxin", was believed to cause myosltis but it was further indicated that this poly-peptide had not been isolated although it appeared to be a polypeptide of approximately 7,000 molecular weight, had a strong net positive of approximately 7,000 molecular weight, had a strong net positive charge and was retained on CM-Sephadex~ at a pH of 8Ø
In the publication "Nature", 247, 11, January 4, 1975 there are described products indentified as Thymin I and Thymin II which were found to be new polypeptides isolated from bovine thymus which have particular uses in various therapeutic areas.
Because of the use of similar names for other products isolated from the thymus in the prior art, these Thymin I and Thymln II
products are now named as Thymopoietin I and Tnymopoietin II.
These products and processes are described in United States Patent 4,077,949 issued March 7, 1978.
In issued United States Patent 4,002,602 dated January 11, 1977, there are disclosed long chain polypeptieds described in Ubiquitous Immunopoietic Polypeptide (UBIP). This peptide has subsequently been renamed as Ubiquitin. This polypeptide is a 74-amino acid polypeptide characterized by its ability to induce in vitro, in nanogram concentrations, the differentiation ,: .. : , . .
.
. .:

1iLg} 5~23 of both T-cell and B-cell immunocytes from precursors present in bone marrow or spleen. Thus, the polypeptide is useful in therapeutic areas involving thymic or immunity deficiencies and the like.
In issued U,S, Patent No. 4,002,740, dated January ll, 1977 there are disclosed synthesized tridecapeptide compositions which have the capability of inducing the differentiation of T-lymphocytes but not of complement receptor B-lymphocytes.
This polypeptide thus exhibited many of the characteristics of the long chain polypeptides isolated and narned as thymopoietin in above-mentioned ~.S. Pat~nt 4,077,949.
The present invention provides a synthesized ~ive-amino acid polypeptide having a definite active site sequence which has been found to exhibit many of the characteristics of the long chain polypeptide isolated and named as Ubiquitous Immunopoietic Polypeptide (UBIP) in the above publications and U.S. Patent No.
4,002,602, dated January 11, 1977.

SUMMARY OF THE INVENTION
It is accordingly one object of this invention to provide new polypeptides which are important biologically.
A further object of the invention is to provide new polypeptides which have the ability in nanogram concentrations to induce differentiation of both T-precursor cells as well as B-precursor cells and are thereby highly useful in the immunity systems of humans and animals.
A further object of the invention is to provide novel intermediate products, methods for synthesizing the novel poly-peptides of this invention, as well as compositions and methods for use in biological actions.

~5g2l3 Other objects and advantages of the invention will become apparent as the description thereof proceeds.
In satisfaction of the foregoing objects and advantages there are provided by this invention novel polypeptides having the following sequence as the active site:
-X-Y-Z-GLN-LYS-wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA.
There are also provided novel polypeptide-resin intermediates formed in the preparation of the polypeptide of this invention which intermediate has the following sequence:

~ -R3-X-Y-Z-GLN-LYS-Resin as well as this peptide intermediate freed from the resin and other protecting groups, wherein X, Y and Z are as above, and Rl, R2, and R3 represent protecting groups on the amino acids indicated if such groups are necessary, and the resin is a solid phase polymer which acts as a support for the reaction. Also provided is a procedure for preparation of the polypeptide of the invention by solid phase peptide synthesis, as well as therapeutic compositions containing the polypeptide, and methods for administration of the polypeptide to humans and animals for effecting biological actions thereon.

DESCRIPTION OF PREFERRED EMBODIMENTS
As indicated above, this invention is concerned with new polypeptides having therapeutic value in various areas, intermediates formed in the preparation of the polypeptides, therapeutic compositions and methods for their use utilizing the polypeptides of this invention, and methods for manufacture of the polypeptides.

In the main embodiment of the present invention, there are provided polypeptides which have the following amino acid sequence as the active site:

Sg23 I. X-Y-Z-GLN-LYS
wherein X is TYR or ALA, Y is ASN or ALA and Z is ILE or ALA, with X being TYR, Y being ASN and Z being ILE, especially preferred.
In a further embodiment, there are provided pentapeptides containing the above mentioned sequence as the active site and which may be described by the following general formula:
II. R-NH-X-Y-Z-GLN-LYS-COR' wherein X, Y and Z are as described above, and R and R' are substituents on the pentapeptide sequence which do not substantially affect the biological activity of the basic active sequence. By this statement is meant that the terminal amino acids on this pentapeptide chain may be modified without departing from the scope of the invention when functional groups or derivatives (R and R') are placed on these terminal amino acids without substantially affecting the biological activity of the molecule. Thus it is to be understood that the terminal amino and carboxylic acid groups are not essential to the biological activity of the penta-peptide as in some polypeptides. Therefore, it is considered that the scope of the present invention is inclusive of these penta-peptides which are terminally unsubstituted and which are terminally substituted by one or more functional groups which do not substantially affect the biological activity disclosed herein.
From this statement it will be understood that these functional groups include such normal substitution as acylation on the free amino group and amidation on the free carboxylic acid group, as well as the substitution of additional amino acids and polypeptides. In these aspects the pentapeptides of this invention appear to be unique since the pentapeptides exhibit the same biological activity as long chain natural peptides in which this pentapeptide sequence forms a portion or occurs therein. It is believed therefore that the activity requirements of the molecule are generated by stereochemistry of the molecule, that is, the particular "folding" of the molecule. In this regard, it should be understood that polypeptide bonds are not rigid but flexible, and may exist as sheets, helices, and the like. As a result, the entire molecule is flexible and will "fold" in a certain way. In the present invention it has been discovered that the pentapeptide "folds" in the same manner as the long chain natural polypeptide and therefore exhibits the same biological characteristics. For this reason, the pentapeptide may be substituted by various functional groups so long as the substituents do not substantially affect the biological activity or interfere with the natural "folds"
of the molecule.
The ability of the molecule to retain its biological activity and natural folding is clearly illustrated by the fact that the pentapeptide sequence of this invention exhibits the same biological characteristics as the natura~l seventy-four amino acid peptide disclosed as Ubiquitin in Patent No. 4,002,602. In this long chain polypeptide, the pentapeptide of this invention may be identified within the molecule but only in combination with the other amino acids described therein. However, this patent is direct evidence that the pentapeptide of this invention is the active site since the biological activities are the same and the amino acids and peptide chains substituted on the terminal amino acids do not affect the biological characteristics of the basic pentapeptide fragment.

In view of this discussion therefore, it will be understood that R and R' in formula II can be any substituent that does not substantially affect the biological activity of the basic active sequence. Thus, for purposes of illustra-tion R and R' may be any of the following substituents:
R R' Hydrogen OH

Cl-C7 alkyl NH2 C6-C20 alkaryl ( 7)2 C6-C20 aralkyl 7 Cl-C7 alkanoyl C2-C7 alkenyl C2-C7 alkynyl wherein R7 is Cl-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C6-C20 aryl, C6-C20 aralkyl or C6-C20 alkaryl, As pointed out above however, R and R' can also be neutral amino acid groups or residues of polypeptide chains having 1 to 20 carbon atoms. The following are illustrative:
R R' ASP GLU
SER SER
LEU THR
SER-ASP LEU
LEU-SER-ASP HIS
LEU-ASP VAL
LEU-SER ARG
GLU-SER

h ~S9~3 R' GLU-THR
GLU-SER-LEU
GLU-SER-THR
GLU-SER-THR-LEU
GLU-SER-THR-LEU-HIS
GLU-SER-THR-LEU-HIS-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG
In a more specific embodiment of the invention, there are provided novel pentapeptides having the following sequence:
III. R-HN-TYR-ASN-ILE-GLN-LYS-COR' wherein R is hydrogen, Cl-C7 alkyl, e.g. methyl, ethyl, C5-C12 aryl, e.g. phenyl, or Cl to C7 alkanoyl, e.g. acetyl or propionyl and R' is OH, NH2, NHR7, N(R7)2. The most preferred polypeptides are those wherein R is hydrogen and R' is OH.
Also included within the scope of the invention are the pharmaceutically acceptable salts of the pentapeptides.
As acids which are able to form salts with the pentapeptides, there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc.
and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, , , -- 1 0 592'3 maleic acid, fumaric acid, anthranylic acid, cinnamic acid, naphthalenesulfonic acid or sulfanylic acid, for instance.
In the above structure the amino acid components of the peptide are identified by abbreviations for convenience.
These abbreviations are as follows:
Amino Acid Abbreviated Designation L-Tyrosine TYR
L-Asparagine ASN
L-Aspartic acid ASP
L-Isoleucine ILE
L-Serine SER
L-Glutamine GLN
L-Leucine LEU
L-Lysine LYS
L-Glutamic GLU
L-Threonine THR
L-Histidine HIS
L-Valine VAL
L-Arginine ARG
L-Alananine ALA
The polypeptides of this invention are five-amino acid peptides which have been found to exhibit characteristics similar to the 74 amino acid polypeptide Ubiquitin (or UBIP) isolated from bovine thymus as disclosed in Patent No. 4,002,602. The peptides of this invention are particularly characterized in their ability to induce the selective differentiation of T-precursor cells as well as B-precursor cells in nanogram concentrations.
It has been found that the polypeptides of this invention induce the differentiation of immunocyte-precursor cells in vitro in the same way as the long chain polypeptides disclosed and BS~Z3 described in Patent No. 4,002,602. Thus, the polypeptides of this invention, even in nanogram concentrations, have been found to induce the differentiation of both T-precursor cells as measured by the acquisition of the thymic differentiation antigens TL and THY-l (~), as well as B-precursor cells as measured by the acquisition of receptors for complement, a distinctive marker of B cells.
To provide an understanding of the importance of the differentiating biological characteristics of the polypeptides of this invention, it should be noted that the function of the thymus in relation to immunity may be broadly stated as the production of thymus-derived cells, or lymphocytes, which are called T cells.
T cells form a large proportion of the pool of recirculating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homograft responses), as effector cells. T cells, however, do not secrete humoral antibodies as these antibodies are secreted by cells derived directly from the bone marrow independently of the thymic influence and these latter cells are termed B cells.
However, for many antigens, B cells require the presence of appropriately reactive T cells before they can produce antibodies.
The mechanism of this process of cell cooperation is not yet completely understood.
From this explanation, it may be said that in operational terms, the thymus is necessary for the development of cellular immunity and many humoral antibody responses and it affects these systems by inducing, within the thymus, the differentiation of haemopoietic stem cells to T cells. This inductive influence is mediated by secretions of the epithelial cells of the thymus, that is, the thymic hormones.

~1~59;~3 Further, to understand the operation of the thymus and the cell system of lymphocytes, and the circulation of lymphocytes in the body, it should be pointed out that stem cells arise in the bone marrow and reach the thymus by the blood stream. Within the thymus, stem cells become differentiated to immunologically competent T cells, which migrate to the blood stream and together with B cells, circulate between the tissues, lymphatics, and the blood stream.
The cells of the body which secrete antibody also develop from hemopoietic stem cells but their differentiation is not determined by the thymus. Hence, they are termed bone marrow-derived cells or B cells. In birds they are differentiated in an organ analogous to the thymus, which is called the Bursa of Fabricius. In mammals no equivalent organ has been discovered and it is thought that B cells differentiate within the bone marrow. The physiological substances dictating this differentiation remain completely unknown.
As pointed out above, the polypeptides of this invention are therapeutically useful in the treatment of humans and animals.
Since the new polypeptides have the capability of inducing the differentiation of lymphopoietic stem cells originating in the hemopoietic tissues to thymus-derived cells or T cells which are capable of involvement in the immune response of the body and also of inducing the differentiation of B cells, the products of this invention are considered to have multiple therapeutic uses.
Primarily, since the compounds have the capability of carrying out certain of the indicated functions of the thymus they have application in various thymic function and immunity areas. A primary field of 11~5923 application is in the treatment of DiGeorge Syndrome, a condition in which there is a congenital absence of thymus. Injection of the polypeptides will overcome this deficiency. Another application is in agammaglobulinemia which is due to a defect of the putative s cell differentiative hormone of the body.
Injection of the polypeptides will overcome this defect. Because of its biological characteristics, the polypeptides being extremely active at low concentrations, are useful in assisting the collective immunity of the body in that they increase or assist in therapeutic stimulation of cellular immunity and humoral immunity and thereby become useful in the treatment of diseases involving chronic infection in vivo, such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like. Further, the peptides are considered to be useful in any area in which cellular or humoral immunity is an issue and particularly where there are deficiencies in immunity such as in the DiGeorge Syndrome mentioned above. Further, because of the characteristics of the polypeptides, they have in vitro usefulness in inducing the development of surface antigens of T cells, in inducing the development of the functional capacity to achieve responsiveness to mitogens and antigens and cell collaborativity in enhancing the ability of B cells to produce antibodies. They have ln vitro usefulness in inducing the development of B cells as measured by the development of surface receptors for complement.
The peptides are also useful in inhibiting the uncontrolled proliferation of lymphocytes which are responsive to Ubiquitin.
An important characteristic of the polypeptide is its in vivo ability to restore cells with the characteristics of T cells and also its in vivo ability to restore cells with the characteristics of B cells.

59~:3 A further important property of the peptides of this invention are that they are highly active in very low concentrations.
Thus, it has been found that the peptides are active in concentrations ranging from 10 nanogram per ml, and are maximally active at concentrations from about 0.05-1 microgram per ml.
The carrier may be any of the well known carriers for this purpose including normal saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent adsorptive losses to glassware at these low concentrations. The peptides of this invention are active at a range of above about 0.1 mg/kg of body weight. For the treatment of DiGeorge Syndrome, the poly-peptides may be administered at a rate of about 0.1 to 10 mg/kg of body weight. Generally, the same range of dosage amounts may be used in treatment of the other conditions or diseases mentioned.
The polypeptides of this invention were prepared using the concepts similar to those described by Merrifield as reported in Journal of American Chemical Society, 85, pps. 2149-2154, 1963.
The synthesis involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by covalent bonds to a solid resin particle. By this procedure, reagents and by-products were removed by filtration and the recrystallization of intermediates were eliminated. The general concept of this method depends on attachment of the first amino acid of the chain to a solid polymer by a covalent bond and the addition of the succeeding amino acids one at a time in a stepwise manner until the desired sequence is assembled. Finally the peptide is removed from the solid support and protective groups removed. This method provides a growing peptide chain attached to a completely insoluble solid particle so that it is in a convenient form to be filtered and washed free of reagents and by-products.

~S~23 The amino acids may be attached to any suitable polymer which merely has to be insoluble in the solvents used and have a stable physical form permitting ready filtration. It must contain a functional group to which the first protected amino acid can be firmly linked by a covalent bond. Various polymers are suitable for this purpose such as cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene but in the synthesis of this invention, there was used a chloromethylated copolymer of styrene and divinylbenzene.
The various functional groups on the amino acid which were active but which were not to enter into the reactions were protected by conventional protecting groups as used in the polypeptide art throughout the reaction. Thus the functional groups on tyrosine and lysine were protected by protecting groups which could be removed on completion of the sequence without adversely affecting the polypeptide final product. The synthesis was performed by a modification of the solid synthesis method in that fluorescamine was used to determine if coupling was complete by an indication of positive fluorescence (see Felix et al, Analyt, Biochem., 52, 377, 1973). If complete coupling was not indicated, the coupling was repeated with the same protected amino acid before deprotection.
The general procedure involved initially esterifying L-lysine, protected on its amino groups, to the resin in absolute alcohol containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and dried. The protecting group on the a-amino group of the lysine amino acid (e.g. t-BOC, i.e., t -butyloxycarbonyl), was then removed without affecting other protecting groups. The resulting coupled amino acid resin, having 5i923 the free amino group, was then reacted with a protected L-glutamine, preferably alpha-tBOC-L-glutamine to couple the L-glutamine. The reactions were then repeated with protected L-isoleucine, L-asparagine and L-tyrosine until the complete molecule was prepared.
The sequence of reactions was carried out as follows:

Resin lR2 1~_BOC_LYS-COOH

~2 ~-BOC-Lys-Resin Remove ~-amino protecting group lR2 H2N-Lys-Resin ~-BOC-L-Gln , IR2 ~-BOC-Gln-Lys-Resin ¦ Remove ~-amino protecting group ~lR2 H2N-Gln-Lys-Resin ¦ ~-BOC-Ile-COOH
~1 lR2 ~-BOC-Ile-Gln-Lys-Resin ¦ Remove ~-amino protecting group J, IR2 H2N-Ile-Gln-Lys-Resin ¦ ~-BOC-Asn-COOH
~, R2 ~-BOC-Asn-Ile-Gln-Lys-Resin ¦ Remove ~-amino protecting group ~ IR2 H2N-Asn-Ile-Gln-Lys-Resin Rl C~-R3-Tyr-COOH

~-R3-Tyr-Asn-Ile-Gln-Lys-Resin ~ Remove all protecting groups H2N-Tyr-Asn-Ile-Gln-Lys-COOH

In the above sequence of reactions Rl and R2 are protecting groups on the various reactive side chains on the amino acids which are not affected or removed when the protective group on the a-amino group is removed to permit further reaction and a-R3 is a protecting group of the a-amino group. Preferably in the above intermediate pentapep-tide resin, the term Rl stands for a protective grouping such as 0-2,6-dichlorobenzyl, R2 stands for ~-2-chlorobenzyloxy-carbonyl and R3 stands for t-butyloxycarbonyl. The resin is any of the resins mentioned above as being useful in the process. In the above series of reactions, the peptides where ALA is substituted for TYR, ASN, or ILE, are prepared in the same manner.
After the final intermediate is prepared, the peptide-resin is cleaved to remove the Rl, R2 and R3 protecting groups thereon and the resin. The protecting groups are removed by conventional means, e.g., by treat-ment with anhydrous hydrogen fluoride, and the resulting free peptide was then recovered.
While the preferred method for production of the polypeptides of this invention is by the use of an insoluble solid polymer as described in the method of Merrifield, it is also to be understood that other methods for preparation may also be used. For example, a different solid support may be employed such as an N-methyl-benzhydrylamine resin or benzhydrylamine resin, which is advantageous under certain conditions. In this procedure the C-terminal amino acid is attached directly to the resin and the finished peptide maybe cleaved from the substrate in HF to form C-terminal amides.

,.,_ " ~."

.. .. .

S~23 Techniques for use of an N-methyl-benzhydrylamine resin are described by Monahan et al in Biochemical and Biophysical Research Communications, _, 1100-1105 (1972). TechniqueS for use of a benzhydrylamine resin are described by J. Rivier, et al, in Journal of Medicinal Chemistry, 1973, vol. 16, 545-549.
Various derivatives of the basic pentapeptide may also be produced using methods known to the art. Obviously, in the production of such derivatives it will be necessary to block functional groups which might interfere with the reaction sequence in order to produce the desired product. For example, the alpha-carboxylic acid group of aspartic acid or the alpha-amino group of lysine should be blocked during preparation of these derivatives.
As pointed out above, in conducting the process it is necessary to protect or block the amino groups in order to control the reaction and obtain the products desired. Suitable amino-protecting groups which may be usefully employed include salt formation for protecting strongly-basic amino groups, or urethane protecting substitutes such as benzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to utilize tert-butyloxy-carbonyl (tBOC) or t-amyloxycarbonyl (AOC) for protecting the a-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since the BOC and AOC (t-amyloxy-carbonyl) protecting groups are readily removed following such reaction and prior to the subsequent step (wherein such a-amino group itself undergoes reaction) by relatively mild action of acids (e.g., trifluoroacetic acid) which treatment does not otherwise affect groups used to protect other reactive side chains. It will thus be understood that the a-amino ~5~23 groups may be protected by reaction with any material which will protect the amino groups for the subsequent reaction(s) but which may later be removed under conditions which will not otherwise affect the molecule. Illustrative of such materials are organic carboxylic acid derivatives which will acylate the amino group.
In general, any of the amino groups can be protected by reaction with a compound containing a grouping of the formula:

1l R - O - C -wherein R is any grouping which will prevent the amino group from entering into subsequent coupling reactions and which can be removed without destruction of the molecule. Thus R is a straight or branched chain alkyl which may be unsaturated, preferably of 1 to 10 carbon atoms, aryl, preferably of 6 to 15 carbons, cycloalkyl, preferably of 5 to 8 carbon atoms; aralkyl, preferably of 7 to 18 carbon atoms, alkaryl, preferably of 7 to 18 carbon atoms, or hetercyclic, e.g., isonicotinyl. The aryl, aralkyl and alkaryl moieties may also be further substituted as by one or more alkyl groups of 1 to about 4 carbon atoms. Preferred groupings for R
include tertiary-butyl, tertiary-amyl, phenyl, tolyl, xylyl and benzyl. Highly preferred specific amino-protecting groups include benzyloxycarbonyl; substituted benzyloxycarbonyl wherein the phenyl ring is substituted by one or more halogens, e.g., Cl or Br; nitro;
lower alkoxy, e.g., methoxy; lower alkyl; tertiary-butyloxycarbonyl, tertiary-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl;
adamantyloxycarbonyl; biphenylisopropoxycarbonyl; and the like.
Other protecting groups which can be used include isonicotinyloxy-carbonyl, phthaloyl, para-tolylsulfonyl, formyl and the like.

~S923 In conducting the general process of the invention, the peptide is built by reaction of the free ~-amino group with a compound possessing protected amino groups. For reaction or coupling, the compound being attacked is activated at its carboxyl group so that the carboxyl group can then react with the free ~-amino group on the attached peptide chain. To achieve activation the carboxyl group can be converted to any reactive group such as an ester, anhydride, azide, acid chloride, or the like.
It should also be understood that during these reactions,the amino acid moieties contain both amino groups and carboxyl groups and usually one grouping enters into the reaction while the other is protected. Prior to the coupling step. the protecting group on the alpha or terminal amino group of the attacked peptide is removed under conditions which will not substantially affect other protecting groups, e.g., the group on epsilon-amino of the lysine molecule. The preferred procedure for effecting this step is mild acidolysis, as by reaction at room temperature with trifluroacetic acid.
As may be appreciated, the above-described series of process steps results in the production of the specific pentapeptide in the following formula:

This pentapeptide also includes the basic active-site sequence of the polypeptide of this invention. The substituted pentapeptide of Formula II, wherein the terminal TYR and LYS amino acid groups may be further substituted as described above, are then prepared by reaction of this basic pentapeptide with suitable reagents to prepare the desired derivatives. Reactions of this type such as acylation, esterification, amidation and the like, are of course well known in the art. Further, other amino acids, that is amino acid groups which do not affect the biological activity of the basic pentapeptide molecule, are added to the peptide chain by the same sequence of reactions by which the pentapeptide was synthesized to either end of the peptide chain.
The following examples are presented to illustrate the invention but it is not to be considered as limited thereto.
In the Examples and throughout the specification, parts are by weight unless otherwise indicated.

EXAMPLE I
In preparation of the polypeptide of this invention the following materials were purchased commercially.
Alpha-BOC-L-Glutamine-O-nitrophenyl-ester Alpha-BOC-~-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-asparagine Alpha-BOC-L-Isoleucine Alpha-BOC-0-2,6-dichlorobenzyl-I,-tyrosine In these reagents, BOC is t-butyloxycarbonyl. "Sequenal"
grade reagents for amino acid sequence determinations, dicyclohexyl carbodiimide, fluorescamine, and the resin were also purchased commercially. The resin used was a polystyrene divinyl benzene resin, 200-400 mesh size containing 1% divinyl benzene and .75 mM
of chloride per gram of resin.
In preparation of the polypeptide, 2m moles of ~-BOC-~-2-chlorobenzyloxycarbonyl-L-lysine were esterified to 2m moles of chloromethylated resin in absolute alcohol containing lmM
triethylamine for 24 hours at 80C. The resulting coupled amino acid resin was filtered, washed with absolute alcohol and dried.

5~3!Z3 Thereafter~ the other ~-BOC amino acids were similarly coupled to the deprotected ~-amino group of the peptide-resin in the correct sequence to result in the polypeptide of this invention using equivalent amounts of dicyclohexyl carbodiimide except for ~-BOC-L-glutamine-O-nitrophenyl ester which was coupled directly. After each coupling reaction, an aliquot of resin was tested with fluorescamine and if positive fluorescence was found, coupling was taken to be incomple-te and was repeated with the same protective amino acid. As a result of the several coupling reactions, the intermediate pentapeptide-resin was prepared.
This peptide-resin was cleaved and the protective groups removed in a Kelf cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0C. for 60 minutes with 1.2 ml anisole per gram peptide-resin as scavenger. The peptide mixture was lyophilized and washed with anhydrous ether and the peptide was chromatographed on P-6 Bio-Gel in 1 N acetic acid. The resulting polypeptide was determined to be 94% pure and was determined to have the following sequence.

EXAMPLE II
To determine the activity and characteristics of the polypeptide, determinations were carried out on healthy 5-6 week old nu/nu mice of both sexes, the mice being bred on a BALBtc background (thymocytes expressing Thy-1.2 surface antigen) and maintained under conventional conditions. For the antisera, anti Thy-1.2 sera were prepared in Thy-l congenic mice.
For the induction of in vitro of Thy-l T cell or -CR B cell differentiation, the induction of thymocyte :

~5923 differentiation from prothymocytes ln vltro was performed as described by Komuro and Boyse, (Lancet, 1, 740, 1973~, using the acquisition of Thy-1.2 as a marker of T cell differentiation.
The induction of CR B cell differentiation from CR B cell precursors _ vitro was performed under similar conditions using as the assay criterion, the capacity of CR s cells to bind sheep erythrocytes coated with subagglutinating quantities of rabbit antibody and nonlytic complement. Spleen cell populations from health nu/nu mice fractionated on discontinuous bovine serum albumin gradients were used as the source of both precursor types (Thy-l and CR ) because they have very few or no Thy-l cells and low numbers of CR cells.
As a result of this determination it was found that the polypeptide displayed a selectivity of actions similar to that of Ubiquitin in inducing the differentiation of T-lymphocytes and of complement receptors (CR+) B-lymphocytes.
The pentapeptide induced differentiation of Thy-l+ T cells in concentrations ranging from lOng to 1 ~g/ml, and also induced ,~
the differentiation of CR B cells in concentrations of 10 ng to 1 ~g/ml.

EXAMPLES III and IV
Example III
The pentapeptide prepared as in Example I, while still coupled to the resin, is treated with trifluoroacetic acid in dichloromethane to remove the t-BOC protecting group from the tryosine moiety. The resulting peptide is then acylated with acetic anhydride, followed by cleavage from the resin substxate and removal of all protective groups with HF.
The following acrylated derivative is thus produced:
A. CH3CONH-TYR-ASN-ILE-GLN-LYS-COOH
Example IV
To produce the amidated derivative of the pentapep-tide of Example I, the protected pentapeptide is Prepared using a benzhydrylamine resin as the substrate, following the method of J. Rivier, et al, referred to above. The amidated deriva-tive is produced by cleaving the protected pentapeptide attached to the basic resin by reaction with hydrogen fluoride. The amidated pentapeptide has the following formula:
B. H2N-TYR-ASN-ILE-GLN-LYS-CONH2 For identification, thin layer chromatography and electrophoresis were employed which provided the following data:
Thin Layer Chromatography:
Sample: 30 ~g Silica Gel (Brinkman glass plate, 5x20cm, 0.25mm thickness) Rl : n-BuOH : Pyridine : HOAc : H2O 30 : 15 : 3 : 12 Rf : EtOAc : Pyridine : HOAc : H2O 5 : 5 : 1 : 3 R : EtOAc : n-BuOH HOAc : H2O 1 : 1 : 1 :
f Spray reagent: Pauly, Ninhydrin & I2 .

~l~iS~23 TLC Electrophoresis R R2 R3 Peptide moved Compounds: f f f to cathode Ac-Tyr-Asn-Ile-Gln-Lys 0.47 0.87 0.54 - 1.35 cm Tyr-Asn-Ile-Gln-Lys-NH2 0.48 0.87 0.37 - 6.60 cm Electrophoresis:
Whatman 3 mm paper (11.5cm x 56.5cm) Sample : 100 ~g pH 5.6, Pyridine-Acetate buffer solution 1000 V, 1 hour Spray reagent: Pauly & Ninhydrin The acetylated pentapeptide of Example III, when utilized in a concentration of 1 ~g/ml in 14% Twomey solution, showed maximum and minimum activities comparable to the basic pentapeptide of Example I.
The amidated pentapeptide of Example IV, when utilized in a concentration of 1 ~g/ml in 6~ Twomey solution, showed activity comparable to the basic pentapeptide of Example I.
EXAMPLE V
The basic pentapeptide prepared according to Example I, while still attached to the resin, is cleaved from the resin by transesterification with sodium methoxide in methanol under transesterification conditions. Removal of the protecting groups yields the esterified product of the following formula:

EXAMPLE VI
The diethylamine derivative of the pentapeptide prepared as in Example I is produced from the methyl ester produced in Example V. In this reaction the methyl ester of Lgl 5~23 Example V is reacted with diethylamine in dimethylformamide solution to produce the following diamino-substituted amide:
H2N-TyR-AsN-ILE-GLN-Lys-coN(c2H5)2 This diethylamine derivative can also be prepared by reaction of the basic pentapeptide of Example I, while still attached to the resin substrate, by cleavage of the peptide from the resin by reaction with diethylamine. The resulting product after removal from the resin and removal of protecting groups is the diethylamide derivative.
EXAMPLE VII
In this example the N-ethyl tyrosine derivative of the basic pentapeptide of Example I is prepared by reaction with ethyl bromide. To carry out the reaction, the alpha-amino group on the lysine moiety remains blocked by ~-2-chloro-benzyloxycarbonyl group but the TYR terminal amino group is freed by reaction with trifluoroacetic acid in dichloromethane.
The blocked intermediate is then reacted with a stoichiometric amount of ethyl bromide under alkylation conditions. The pro-tective group is then removed from the LYS alpha-amino acid to -form the substituted polypeptide of the following formula:

EXAMPLE VIII
In this example, the amidated derivative of the ethylamino polypeptide of Example VII is produced. The reactions of Example VII are carried out while permitting the basic pentapeptide to remain coupled to the resin sub-strate and the N-ethyl derivative is produced without cleav-ing from the substrate. Thereafter, this intermediate product attached to the resin substrate is cleaved from the resin with ';' anhydrous ammonia in dimethylformamide solvent to form the amido polypeptide of the following formula:

EXAMPLE IX
In this example, the acylated pentapeptide prepared as in Example III, while still attached to the resin substrate, is cleaved by reaction with anhydrous ammonia under amidation conditions to free the compound and, following removal of the protecting groups, form the following polypeptide:

EXAMPLES X-XXI
Using the reaction techniques described hereinabove for the lengthening of the polypeptide chain, the following polypeptides are prepared which contain the active amino acid sequence but which are substituted on the terminal amino and carboxylic groups by R and R' to provide the basic amino acid of the formula:
R-NH-TYR-ASN-ILE-GLN-LYS-COR' which is substituted by the amino acids given in the following Table as indicated.
EXAMPLE NO. _ R' X ASP OH
XI SER-ASP OH
XII LEU-SER-ASP OH
XIII LEU-SER-ASP GLU
XIV LEU-SER-ASP GLU-SER
XV LEU-SER-ASP GLU-SER-THR
XVI LEU-SER-ASP GLU-SER-THR-LEU
XVII LEU-SER-ASP GLU-SER-THR-LEU-HIS

~5~23 EXAMPLE NO. R R' XVIII LEU-SER-ASP GLu-sER-THR-LEu-HIs-LEu XIX ASP GLU
XX ASP GLU-SER
XXI LEU-SER-ASP GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG
The polypeptide derivatives prepared in Examples V-XXI retain the biological activity as described herein for the basic amino acid sequence.
EXAMPLES XXII, XXIII and XXIV
Using the sequencing procedure described for Example I, the followingpentapeptides are prepared:
Ex. XXII H2N-ALA-ASN-ILE-GLN-LYS-COOH
Ex. XXIII H2N-TYR-ALA-ILE-GLN-LYS-COOH
: Ex. XXIV H2N-TYR-ASN-ALA-GLN-LYS COOH.
To determine the pharmacological activity and characteristics of these pentapeptides, use was made of the induction of Th-l (T-cell) antigen on chicken bone marrow for use as an assay of the peptides at a concentration of 1 ~g/mil.
This method is described in Science, Brand et al, Vol. 193, pp. 319-321, July, 1976. In this method pooled cells from femur and tibiotarsus of newly hatched chicks of strain SC
(Hy-Line) are fractionated by ultracentrifugation on a five-layer discontinuous bovine serum albumin (BSA) gradient (11).
Cells from each interface are washed and suspended for incubation at a concentration of 5 x 106 cells per milliliter with the peptide (0.1 ~g/ml) in RPMI 1630 medium supplemented with 15 mM
Hepes, 5 percent y-globulin-free fetal calf serum, deoxyribonu-clease (14 to 18 unit/ml), heparin (5 unit/ml), penicillin (100 unit/ml), and streptomycin (100 ~g/ml). Controls are 1.,~, ~l~S923 incubated with BSA (l ~g/ml) or medium alone. After incubation, the cells are tested in the cytotoxicity assay using chicken and guinea pig complement fractions. The proportion of Bu-l or Th-l cells in each layer are calculated as a cytotoxicity index, lO0 (a-b)/a, where a and b are the percentages of viable cells in the complement control and test preparation, respec-tively. The percentage of cells induced is obtained by sub-tracting the mean values in the control incubations without inducing agents (usually l to 3 percent) from those of the test inductions.
As a result of the characterizations of this type, the pentapeptides of Examples XXII, XXIII and XXIV were found to show the following percentages of inductions of Th-l (T-cell) antigen on chicken bone marrow.
Example No. ~ Induction XXII 12%
XXIII 15%
XXIV 21%
The invention has been described herein with reference to certain preferred embodiments. However, as obvious variations will appear to those skilled in the art, the invention is not to be considered as limited thereto.

, :

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for manufacture of an active peptide fragment which has the capability of inducing differentiation of both T-lymphocytes and complement receptor (CR+) B-lymphocytes, which has the following active group therein:
R-NH-X-Y-Z-GLN-LYS-COR' wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA, and R and R' are terminal groups on said polypeptide which do not substantially affect the biological capability thereof, wherein R and R' are selected from the groups consisting of:

wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C6-C20 aryl, C6-C20 aralkyl, or C6-C20 alkaryl, which comprises esterifying L-lysine protected on its amino groups, to an insoluble resin polymer by covalent bonding;
removing the .alpha.-amino protecting group from the L-lysine moiety, reacting with an .alpha.-amino protected L-glutamine to couple L-glutamine to the L-lysine resin; removing the .alpha.-amino protect-ing group from the L-glutamine moiety, reacting with an .alpha.-amino protected L-isoleucine or L- alanine to couple L- isoleucine or L-alanine to the L-glutamine-L-lysine-resin; removing the a-amino protecting group and reacting with an a-amino protected L-asparagine or L-alanine to couple L-asparagine or L-alanine to the L-isoleucine or L-alanine-L-glutamine-L-lysine-resin, removing the a-amino protecting group and reacting with an a-amino protected L-tyrosine or L-alanine to couple L-tyrosine or L-alanine to L-asparagine or L-alanine-L-isoleucine or L-alanine-L-glutamine-L-lysine-resin, removing all amino-protecting groups from the peptide, separating from the resin polymer, and reacting the resulting pentapeptide with an appro-priate reagent so as to form the derivatives identified by R
and R' with protection of unreactive functional groups, if necessary.

2. A process according to claim 1 wherein reactive side chains on the reacting amino acids are protected during the reaction.

3. A process according to claim 1 wherein the resin polymer is selected from the group consisting of cellulose, polyvinylalcohol, polymethacrylate, sulfonated polystyrene, and a chloromethylated copolymer of styrene and divinylbenzene.

4. A polypeptide fragment which has the capability of inducing differentiation of both T-lymphocytes and complement receptor (CR+) B-lymphocytes, which has the following active group therein:
R-NH-X-Y-Z-GLN-LYS-COR' wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA, and R and R' are terminal groups on said polypeptide which do not substantially affect the biological capability thereof, wherein R and R' are selected from the groups consisting of:

wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C6-C20 aryl, C6-C20 aralkyl, or C6-C20 alkaryl, whenever produced by the process of claim 1 or its obvious chemical equivalent.

5. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein said polypeptide is a pentapep-tide of the following sequence:
R-NH-TYR-ASN-ILE-GLN-LYS-COR' wherein R is hydrogen, C1-C7 alkyl, C5-C12 aryl, or C1-C7 alkanoyl, and R' is OH, NH2, NHR7 or N(R7)2.

6. A pentapeptide of the following sequence:
R-NH-TYR-ASN-ILE-GLN-LYS-COR' and the pharmaceutically acceptable salts thereof, wherein R
is hydrogen, C1-C7 alkyl, C5-C12 aryl, or C1-C7 alkanoyl, and R' is OH, NH2, NHR7 or N(R7)2, whenever produced by the process of claim 5 or its obvious chemical equivalent.

7. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is hydrogen and R' is OH.

8. A polypeptide according to claim 4 wherein R is hydrogen and R' is OH, whenever produced by the process of claim 7 or its obvious chemical equivalent.

9. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is CH3CO- and R' is OH.

10. A polypeptide according to claim 4 wherein R is CH3CO- and R' is OH, whenever produced by the process of claim 9 or its obvious chemical equivalent.

11. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is CH3 and R' is OH.

12. A polypeptide according to claim 4, wherein R
is CH3 and R' is OH, whenever produced by the process of claim 11 or its obvious chemical equivalent.

13. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is H and R' is NH2.

14. A polypeptide according to claim 4 wherein R is H and R' is NH2, whenever produced by the process of claim 13 or its obvious chemical equivalent.

15. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is H and R' is N(C2H5)2.

16. A polypeptide according to claim 4 wherein R is H and R' is N(C2H5)2, whenever produced by the process of claim 15 or its obvious chemical equivalent.

17. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is CH3CO- and R' is NH2.

18. A polypeptide according to claim 4 wherein R is CH3 CO- and R' is NH2, whenever produced by the process of claim 17 or its obvious chemical equivalent.

19. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is H and R' is -OCH3.

20. A polypeptide according to claim 4 wherein R is H and R' is -OCH3, whenever produced by the process of claim 19 or its obvious chemical equivalent.

21. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is H and R' is OC2H5.

22. A polypeptide according to claim 4 wherein R is H and R' is OC2H5, whenever produced by the process of claim 21 or its obvious chemical equivalent.

23. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is C2H5 and R' is OC2H5.

24. A polypeptide according to claim 4 wherein R is C2H5 and R' is OC2H5, whenever produced by the process of claim 23 or its obvious chemical equivalent.

25. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is CH3 and R' is NH2.

26. A polypeptide according to claim 4 wherein R is CH3 and R' is -NH2, whenever produced by the process of claim 25 or its obvious chemical equivalent.

27. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is ASP and R' is OH.

28. A polypeptide according to claim 4 wherein R is ASP and R ' is OH, whenever produced by the process of claim 27 or its obvious chemical equivalent.

29. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is SER-ASP and R' is OH.

30. A polypeptide according to claim 4 wherein R is SER-ASP and R' is OH, whenever produced by the process of claim 24 or its obvious chemical equivalent.

31. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is LEU-SER-ASP and R' is OH.

32. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is OH, whenever produced by the process of claim 31 or its obvious chemical equivalent.

33. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is LEU-SER-ASP and R' is GLU.

34. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is GLU, whenever produced by the process of claim 33 or its obvious chemical equivalent.

35. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is LEU-SER-ASP and R' is GLU-SER.

36. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is GLU-SER, whenever produced by the process of claim 35 or its obvious chemical equivalent.

37. A process for the manufacture of a polypeptide, as claimed in claim 1 wherein R is LEU-SER-ASP and R' is GLU-SER-THR.

38. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is GLU-SER-THR, whenever produced by the process of claim 37 or its obvious chemical equivalent.

39. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is LEU-SER-ASP and R' is GLU-SER-THR-LEU.

40. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is GLU-SER-THR-LEU, whenever produced by the process of claim 39 or its obvious chemical equivalent.

41. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is LEU-SER-ASP and R' is GLU-SER-THR-LEU-HIS.

42. A polypeptide according to claim 4 wherein R is LEU-SER-ASP and R' is GLU-SER-THR-LEU-HIS, whenever produced by the process of claim 41 or its obvious chemical equivalent.

43. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is ASP and R' is GLU.

44. A polypeptide according to claim 4 wherein R is ASP and R' is GLU, whenever produced by the process of claim 43 or its obvious chemical equivalent.

45. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is ASP and R' is GLU-SER.

46. A polypeptide according to claim 4 wherein R is ASP and R' is GLU-SER, whenever produced by the process of claim 45 or its obvious chemical equivalent.

47. A process for the manufacture of a polypeptide, as claimed in claim 1, wherein R is H and R' is GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG.

48. A polypeptide according to claim 4 wherein R is hydrogen and R' is GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG, whenever produced by the process of claim 47 or its obvious chemical equivalent.