GB1565032A - Polypeptide compositions and methods for their manufacture - Google Patents

Description

(54) IMPROVEMENTS IN OR RELATING TO POLYPEPTIDE COMPOSITIONS AND METHODS FOR THEIR MANUFACTURE (71) We, SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH, a Corporation of the State of New York, of 1275, York Avenue, New York, New York 10021, U.S.A., do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to polypeptides.

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 0.8own of his body weight at birth, although it has been previously hypothesized that a neuromuscular blocking substance existed in the thymus and that thymic hormone affected the development of the immune system. Despite keen interest in possible functions of the thymus and early speculation and experimentation, little was known of the function of the thymus until recently. It is now realized, however, that the thymus is a compound organ with both epithelial (endocrine) and lymphoid (immunological) components and thus the thymus is involved in the immunity functions of the body.

The thymus 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 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.

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 co-operation 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.

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 haemopoietic 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.

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. 23O240, 1971: Clinical and Experimental Immunologv. Vol.4, No. 2, pps. 181--189, 1967: Nature. Vol. 247, pps. 11--14, 1974: Proceedings of the National Academy of Sciences, USA. Vol. 71. pps. 1474-1478, 1974: Cell, Vol. 5, pps. 361-365, and pps. 367-370, 1975; and Lancet, Vol. 2, pps.

256-259, 1975.

In the article by Goldstein and Manganaro in Annals of the New York Facades of Sciences, Vol. 183, pps. 230-240, 1971, there are disclosures regarding the presence of a thymic polypeptide 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 myositis but it was further indicated that this polypeptide had not been isolated although it appeared to be a polypeptide of approximately 7,000 molecular weight, had a strong net positive charge and was retained on CM Sephadex (Registered Trade Mark) at a pH of 8.0.

In the publication "Nature", 247, 11, January 4, 1975, there are described products identified 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 Thymin II products are now named as Thymopoietin I and Thymopoietin II. These products and processes are described in United States Patent No. 4,077,949 filed August 22, 1975.

In issued United States Patent Number 4,002,602, dated January 11, 1977 there are disclosed long chain polypeptides described as Ubiquitous Immunopoietic Polypeptides (UBIP), which polypeptide is a 74-amino acid polypeptide characterized by its ability to induce in vitro, in nanogram concentrations, the differentiation 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.

In issued United States Patent No. 4,002,740, dated January 11, 1977 there are disclosed synthesized tridecapeptide compositions which have the capability of inducing the differentiation of T-lymphocytes but not of complement receptor Blymphocytes. This polypeptide thus exhibited many of the characteristics of the long chain polypeptides isolated and named as thymopoietin in above-mentioned United States Patent No. 4,077,949.

The present invention provides a synthesized 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 Thymopoietin in the above publications and United States Patent No. 4,077.949. filed August 22, 1975, and the tridecapeptide described in U.S. Patent No. 4,002,740.

It is accordingly one object of this invention to provide polypeptides which are important biologically.

A further object of the invention is to provide polypeptides which have the ability in nanogram concentrations to induce differentiation of bone marrow cells to T cells thus giving rise to thymus-derived lymphocytes and thereby are highly useful in the immunity system of humans and animals.

A further object of the invention is to provide intermediate products, methods for synthesizing the polypeptides of this invention, as well as compositions for use in biological actions.

Other objects and advantages of the invention will become apparent as the description thereof proceeds.

According to the present invention a polypeptide having the capability of inducing the differentiation of T-lymphocytes but not of complement receplor (cur) B lymphocytes, said polypeptide has the following sequence: R-ARG-LYS-ASP-VAL-TYR-R' wherein R and R' are terminal groups on said polypeptide which do not substantially affect the biological capability thereof, and the pharmaceutically acceptable salts, and wherein R and R' are independantly selected from: R H OH C1-C7 alkyl NH2 C6-C12 aryl NHR7 C7-C20 alkaryl N(R7)2 C7-C20 aralkyl OR7 C2-C7 alkanoyl C2-C7 alkenyl VAL-OH C2-C7 alkynyl GLN-OH H-GLN LEU-OH H-GLU TYR-OH H-GLY VAL-GLN-OH H-GLU-GLN VAL-LEU-OH H-GLY-GLN VAL-TYR-OH H-GLY-GLU GLN-LEU-OH H-GLY-GLU-GLN GLN-TYR-OH GLN-VAL-OH LEU-TY R-OH LEU-LEU-OH TYR-LEU-OH VAL-GLN-LEU-OH VAL-GLN-LEU-TYR-OH wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl C6-C20 aryl, jC7-C2p aralkyl, or C7-C20 alkaryl.

There are also provided peptide-resin intermediates formed in the preparation of the polypeptides of this invention, which intermediates have the following sequence:

wherein R1, R2, R3, R4 and R5 represent protecting groups on the amino acids indicated, and the Resin is a solid phase polymer which acts as a support for the reaction. Also provided are procedures for preparation of the polypeptides of the invention by solid phase peptide synthesis, as well as therapeutic compositions containing the polypeptides.

As is indicated above the 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 polypeptides of this invention appear to be unique since the polypeptides exhibit the same biological activity as long chain natural peptides in which this polypeptide 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 or helices. 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 polypeptides of the invention "fold" in the same manner as the long chain natural polypeptide and therefore exhibits the same biological characteristics. For this reason, the pentapeptide unit 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 natural forty-nine amino acid disclosed as Thymopoietin in United States Patent No. 4,077,949 of Goldstein and in the publication Nature, 247, 11, 1975. Moreover, the polypeptides of the present invention exhibits the same biological characteristics as the synthesized tridecapeptide disclosed in Cell, Vol. 5, 367-370, August, 1975, and in U.S. Patent No. 4,002,740. In both of these long chain polypeptides, the basic pentapeptide unit of this invention may be identified within the molecule but only in combination with the other amino acids described therein. However, these publications are direct evidence that the polypeptides of this invention contain the active site since the biological activities are the same and the amino acids and peptide chains substituted on the terminal amino acids of the pentapeptide unit do not affect the biological characteristics.

In a more specific embodiment of the invention, there is provided a polypeptide having the following sequence: III. R-ARG-LYS-ASP-VAL-TYR-R' wherein R is hydrogen, C1-C7, alkyl, e.g. methyl, ethyl, C5-C12 aryl, e.g. phenyl, or CX to C7 alkanoyl, e.g. acetyl or propionyl and R' is OH, NH2, NHR7, N(R7)2.

The most preferred polypeptide is that wherein R is hydrogen and R' is OH.

Also included within the scope of the invention are the pharmaceutically acceptable salts of the polypeptides.

As acids which are able to form salts with the pentapeptide, there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranylic acid, cinnamic acid, naphthalenesulfonic acid or sulfanylic acid, for instance.

In the above structures the amino acid components of the peptides are identified as abbreviations for convenience. These abbreviations are as follows: Abbreviated Amino Acid Designation Glycyl GLY L-glutamyl GLU L-glutaminyl GLN L-arginyl ARG L-lysyl LYS L-aspartyl ASP L-valyl VAL L-tyrosyl TYR L-leucyl LEU The polypeptide sequence active site of this invention is a five amino acid peptide which has been found to exhibit characteristics similar to the 49-amino acid polypeptide isolated from bovine thymus as disclosed in United States Patent No.

4,077,949. The peptides of this invention are particularly characterized in their ability to induce the selective differentiation of Thy-l+ T cells (but not CR+ B cells), in concentrations of 1 ng to 10 z,g/ml of carrier solution. Thy-I is a differentiation alloantigen present on T cells but not B cells whereas CR is a complement receptor present on B cells but not T cells.

Studies of these synthetic peptides in the induction assay in vitro showed them to have the same induction specificity as Thympoietin il. That is, they induced the differentiation of Thy-l- cells to Thy-l+ T cells, but did not induce the differentiation of CR' cells to CR+ cells. While many substances have been identified that can mimic Thymopoietin in vitro and induce T cell differentiation by raising intracellular cyclic AMP, it is emphasized that few substances are active at such low concentration, and the peptides of this invention are selective in inducing T cell differentiation but not CR+ B cell differentiation. These synthetic peptides were also shown to affect neuromuscular transmission, like Thymopoietin itself.

Thus, 24 hours after a single injection of l0-l00g per mouse, there was a definite neuromuscular transmission impairment detectable by electromyography.

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 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. Because of their biological characteristics, which are extremely active at low concentrations, they are considered useful in assisting the collective immunity of the body in that the polypeptides will increase or assist in therapeutic stimulation of cellular 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. Further, the compounds are considered to be useful in any area in which cellular immunity is an issue and particularly where there are deficiencies in immunity such as in the DiGeorge Syndrome mentioned above.

Also, where there is an excess of antibody production due to unbalanced T cells and B cells, the compounds can correct this condition by stimulating T cell production. Thus, there may be of therapeutic use in certain autoimmune diseases in which damaging antibodies are present, for example, systemic lupus erythematosus. 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. The polypeptides are also useful in inhibiting the uncontrolled proliferation of Thymopoietin-responsive lymphocytes.

An important characteristic of the polypeptides is their in vivo ability to restore cells with the characteristic of the T cells. Therefore, the polypeptides of this invention are active in many areas as a result of their ability to enhance the immune response in the body. Since the polypeptides of this invention affect neuromuscular transmission, very high doses of the peptides of this invention will be useful in treating diseases with excess neuromuscular transmission, such as spasticity.

A further important property of the peptides of this invention is that they are highly active in very low concentrations ranging from 1 nanogram per ml, and are maximally active at concentrations from about 100 nanogram per ml. The carrier may be any of the well known carriers for this purpose including 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 at least 1 mglkg of body weight. For the treatment of DiGeorge Syndrome, the polypeptides may be administered at a rate of 1.0 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 concepts similar to the method of 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 as 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 protecting groups removed. This method provides a growing peptide chain attached to a completely insoluble solid resin support so that it is in a convenient form to be filtered and washed free of reagents and by-products.

The amino acids may be attached to any polymer which merely is insoluble in the solvents used and has 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, poly(vinyl alcohol), poly(methylmethacrylate) and sulfonate polystyrene but in the synthesis in the Examples, there was used a chloromethylated copolymer of styrene and divinylbenzene.

The various functional groups on the amino acid 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 such as the side chain on arginine, lysine, aspartic acid, and tyrosine 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 tu- amino deprotection.

The general procedure involved initially esterifying L-tyrosine, protected on its amino and hydroxyl groups, to the resin in absolute alcohol containing an amine.

The coupled amino acid resin was then filtered, washed with ethanol and water and dried. The protecting group on the amino group of the protected tyrosine (e.g., tbutyloxycarbonyl, abbreviated as t-BOC), was then removed without affecting other protecting groups. The resulting coupled amino acid-resin, having the free amino group, was then reacted with a protected L-valine, preferable alpha t-BOC L-valine to couple the L-valine to the amino acid-resin. The reactions were then repeated with protected L-aspartic acid, L-lysine and L-arginine until the complete molecule was prepared. This procedure was used in formation of the basic amino acid active sequence. The addition of other neutral amino acid residues on either end of the chain is carried out using the same sequence of reactions as known in the art. The sequence of reactions to prepare the amino acid active site may be carried out as follows:

Resin R4 o-BOCTyrCOOH R4 a-SOC-Tvr-Resin I Ren,,ve airmino amino protecting group A4 H- Tyr-Resin a-BOC- I - Valine * IR4 a-BOC- Val- Tyr-Resin Remove a-amino protecting group R4 H-Val-Tyr-Resin OR3 rr-BOC--Asp-OH Ol R3 R4 a-BO-Asp-Vai-7vrResin Remove aamino protecting group R4 H-Asp-Vat-Tyr-Resin ,R2 a-RO-Lys-OW R2 OjR3 ff4 a-SOC-I ye-Asp-VaI-Tyr-Resin Remove amino protecting group R2 OR3 At4 H-Ivs-Asp-Vai- Tyr-Resin AT cL-AOC-Arg-OH RI R2 tOR3 1 F R4 a-AOC-Arg-I y'A'sp-Vai-Tlr-Re5in I Remove all protecting groups H-Argys-AsP-VaI-7yr-9H In the above sequence of reactions R1, R2, R3, and R4 are protecting groups on the various reactive groups on the amino acid side chains which are not affected or removed when the a-amino protecting group is removed to permit further reaction.

Preferably, in the above intermediate pentapeptide resin, the expression R1 is tosyl (Tos), R2 stands for 2-chlorobenzyloxycarbonyl (C1Z), R3 stands for benzyl (Bzl), R4 stands for 2,6-dichlorobenzyl, and R5 is t-amyloxycarbonyl (tAOC). The resin is any of the resins mentioned above as being useful in the process.

The peptide-resin is cleaved to free the peptide from the resin and protecting groups R1, R2, R2, R4,, and R5 simultaneously to provide the final product polypeptide. The protecting groups and resin were cleaved by conventional means, e.g., by treatment with anhydrous hydrogen fluoride and the peptide recovered.

As pointed out above, in conducting the process it is necessary to protect or block the amine 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 substituents such as benzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to utilise tert-butyloxycarbonyl (tBOC) or t-amyloxycarbonyl (tAOC) for protecting the a-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since the BOC and AOC protecting groups are readily removed following such reaction, and prior to the subsequent step (wherein such a-amino group itself undergdes reaction), by relatively mild action of acids (e.g., trifluoroacetic acid). This treatment does not otherwise affect protecting groups on said chains. It will thus be understood that the a-amino group may be protected by reaction with any material which will protect the a-amino group 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, the amino groups can be protected by reaction with a compound containing a grouping of the formula:

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 heterocyclic, e.g.

isonicotinyl. The aryl, aralkyl, and alkaryl moieties may also be further substituted as by one or more alkyl groups of I to about 4 carbon atoms. Preferred groupings for R include tertiary-butyl, tertiaryl-amyl, phenyl, tolyl, xylyl, and benzyl. Highly preferred specific aminoprotecting groups include benzyloxycarbonyl; substituted benzyloxycarbonyl wherein the phenyl ring is substituted by one or more halogens, e.g., Cl or Br; nitro; C1 to C7 alkoxy, e.g., methoxy; C1 to C7 alkyl; tertiarybutyloxycarbonyl; tertiary-amyloxycarbonyl; cyclohexyloxycarbonyl: vinyloxycarbonyl; adamantyloxycarbonyl; and biphenylisopropoxycarbonyl. Other protecting groups which can be used include isonicotinyloxycarbonyl, phthaloyl, para-toluenesulfonyl and formyl.

In conducting the general process of the invention, the peptide is built by reaction of the free a-amino group of the peptide chain with a compound containing a blocked a-amino group. For reaction or coupling, the carboxyl component of the compound being bonded is activated at its carboxyl group so that the carboxyl group can then react with the free amino group on the peptide chain. To achieve activation, the carboxyl group can be converted to any reactive group such as an ester, anhydride, azide or acid chloride. 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 peptide attached to the resin is removed under conditions which will not substantially affect other protecting groups, e.g., the groups on the epsilon-amino group of the lysine molecule. The preferred procedure for effecting this step is mild acid hydrolysis, as by reaction at room temperature with trifluoroacetic acid.

As may be appreciated, the above-described series of process steps results in the production of the specific polypeptide of the following formula: H-A RG-LYS-ASP-VA L-TYR-OH This polypeptide also includes the basic active-site sequence of the polypeptide of this invention. The sub

Further. other amino acids, that is amino acid groups which do not affect the biological activity of the basic polypeptide molecule, may be added to the peptide chain by the same sequence of reactions by which the polypeptide was synthesized.

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-AOC-Ng-Tos-L-arginine Alpha-BOC-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-L-aspartic acid ss-benzyl ester Alpha-BOC-L-valine Alpha-BOC-2,6-dichlorobenzyl-L-tyrosine In these reagents, BOC is t-butyloxycarbonyl, AOC is t-amyloxycarbonyl, and Tos is tosyl. "Sequenal" grade reagents for amino acid sequence determination, dicyclohexyl carbodiimide, fluorescamine, and the resin were also purchased commercially. The resin used was a polystyrene divinyl benzene resin, 200--400 Tyler mesh size containing 1n by wt. divinyl benzene and 0.75 mM of chloride per gram of resin.

In preparation of the polypeptide, 2 mmoles of -BOC-0-2,6-dichlorobenzyl- L-tyrosine were esterified to 2 mmoles chloromethylated resin in absolute ethanol containing 1 mM triethylamine for 24 hours at 800 C. The resulting coupled amino acid resin was filtered, washed with absolute ethanol and dried. Thereafter, the - BOC or a-AOC 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 dicyclohexylcarbodiimide. After each coupling reaction, an aliquot of resin was tested with fluorescamine and if positive fluorescence was found, coupling was taken to be incomplete and was repeated with the same protected amino acid. As the result of the several coupling reactions, the following pentapeptide-resin resulted:

Tos Z OBzl Bzl(Cl2) l \ l (r-AOC-ARG-LYS-ASP-VAL-TYR-Resin where AOC is amyloxycarbonyl, Tos is tosyl, Z is benzyloxycarbonyl, and Bzl is benzyl, and Bzyl(C12) is 2,6-dichlorobenzyl.

This peptide-resin was cleaved and the protecting groups removed in a Kelf cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 C. for 60 minutes with 1.2 ml anisole per gram peptide-resin as scavenger. The peptide mixture was lyophilized and washed with anhydrous diethyl ether and the peptide extracted with glacial acetic acid and water. The peptide was chromatographed on P-6 Bio-Gel in 1 N acetic acid. The resulting polypeptide was determined to be 940n pure and was determined to have the following sequence: H-A RG-LYS-ASP-VAL-TYR-OH EXAMPLE II To determine the activity and characteristics of the polypeptide, determinations were carried out on healthy 5-6 week nu/nu mice of both sexes, the mice being bred on a BALB-c background (thymocytes expressing Thy-l.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 in vitro of Thy-l+ T cells or CR B cell differentiation, the induction of thymocyte differentiation from prothymocyes in vitro 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 in vitro was performed under similar conditions using as the assay criterion, the capacity of CR" B cells to bind sheep erythrocytes coated with sub-agglutinating quantities of rabbit antibody and nonlytic complement. Spleen cell populations from healthy nu/nu mice fractionated on discontinuous bovine serum albumin gradients were used as source of both precursor tvnes (Thv-1 Clnfl npw L-- Ln the As a result of this determination it was found that the polypeptide displaved a selectivity of actions similar to that of Thymopoietin 11 in inducing the differentiation of T-lymphocytes but not of complement receptors (CR+) Blymphocytes. The pentapeptide induced differentiation of Thv-l+ T cells in concentrations ranging from 1 ng to 10 mg/ml. It did not induce the differentiation of CR B cells in concentrations of 0.01 ng to 10 ,ug/ml.

EXAMPLE III To determine the effect of this peptide on neuromuscular transmission. mice were injected with 10--100 ,ug peptide in N saline intraperitoneally. Twenty-four hours later, neuromuscular transmission was assessed by electromyography as described in Lancet, Vol. 12, pps. 256-259, 1975. There was a detectable neuromuscular impairment in the mice injected with the peptide.

EXAMPLE IV The pentapeptide of Example I was amidated by reaction with ammonia by known methods to form the following derivative: H-A RG-LYS-ASP-VA L-TYR-NH2 For identification, thin layer chromatography and electrophoresis were employed which provided the following data: Thin Layer Chromatography Sample: 30 ,ug Silica gel (Brinkman glass plate, 5x20 cm, 0.25 mm thickness) RI n-BuOH : Pyridine : HOAc H2O 30:15:3:12 R21 : EtOAc : Pyridine : HOAc : H2O 5:5:1:3 R3f EtOAc ' n-BuOH HOAc H2O 1:1:1:1 Spray reagent: Pauly, Ninhydrin, Sakaguchi and I, Electrophoresis TLC Peptide moved Compounds Rf R2t R3f to cathode H-Arg-Lys-Asp-Val-Tyr-NH2 0.53 0.68 0.26 7.5 cm Electrophoresis Whatman (Registered Trade Mark) 3 mm paper (11.5 cm 56 cm) Sample: 100 ,ug pH 5.6, Pyridine acetate buffer solution 1000 V, 1 hour Spray reagent: Ninhydrin and Sakaguchi This amidated polypeptide, when utilized in a concentration of 1 yg/ml in Twomey solution, showed a maximum activity of 105%, and a minimum activity of 70%, when compared with the basic polypeptide of Example 1.

EXAMPLE V The basic polypeptide prepared in Example I is reacted with a stoichiometric amount of ethyl alcohol under esterification conditions and the amino acid of the following formulae is formed and recovered: H-A RG-LYS-ASP-VAL-TYR-OC2H5 EXAMPLE VI The polypeptide prepared as in Example I is reacted with methylamine in stoichiometric amounts under reaction conditions known in the art to prepare the following amino substituted derivative.

H-ARG-LYS-ASP-VAL-TYR-NHCH3 EXAMPLE VII The polypeptide prepared as in Example I is reacted with methyl chloride under alkylation conditions to form the substituted derivative of the following formula: CH3-A RG-LYS-ASP-VAL-TYR-OH EXAMPLE VIII The methyl amino polypeptide prepared as in Example VII is reacted with ammonia in stoichiometric amounts under amidation conditions to form the amido polypeptide of the formula: CH3-ARG-LYS-ASP-VAL-TYR-NH2 EXAMPLE IX The methyl substituted polypeptide prepared as in Example VIII is reacted with ethyl alcohol to esterify the carboxylic group under esterification conditions to form the following esterified polypeptide: CH3-A RG-LYS-ASP-VAL-TYR-OC2H5 EXAMPLES XXIX 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-A RG-LYS-ASP-VA L-TYR-R' which is substituted by the amino acids given in the following Table as indicated.

Example No. R R' X H-GLN OH Xl H-GLU-GLN OH XII H-GLY-GLU-GLN OH XIII H-GLY-GLU-GLN VAL-OH XIV H-GLY-GLU-GLN VAL-GLN-OH XV H-GLY-GLU-GLN VAL-GLN-LEU-OH XVI H-GLY-GLU-GLN VAL-GLN-LEU-TYR-OH XVII H-GLN VAL-OH XVIII H-GLN VAL-GLN-OH XIX H-GLN VAL-GLN-LEU-OH EXAMPLE XX The polypeptide prepared as in Example I was acylated by reaction with acetyl chloride by known methods to prepare the following acylated derivative: CH3CO-ARG-LYS-ASP-VAL-TYR-OH EXAMPLE XXI The polypeptide prepared as in Example XX was also amidated by reaction with ammonia by known methods to prepare the following derivative: CH3CO-ARG-LYS-ASP-VAL-TYR-NH2 The polypeptide derivatives prepared in Examples VXXI retain the biological activity as described herein for the basic amino acid sequence.

WHAT WE CLAIM IS: 1. A polypeptide having the capability of inducing the differentiation of Tlymphocytes but not of complement receptor (CR+) B lymphocytes, said polypeptide having the following sequence: R-ARG-LYS-ASP-VAL-TYR-R' wherein R and R' are terminal groups on said polypeptide which do not substantially affect the biological capability thereof, and the pharmaceutically acceptable salts, and wherein R and R' are independently selected from

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (31)

**WARNING** start of CLMS field may overlap end of DESC **. EXAMPLE VIII The methyl amino polypeptide prepared as in Example VII is reacted with ammonia in stoichiometric amounts under amidation conditions to form the amido polypeptide of the formula: CH3-ARG-LYS-ASP-VAL-TYR-NH2 EXAMPLE IX The methyl substituted polypeptide prepared as in Example VIII is reacted with ethyl alcohol to esterify the carboxylic group under esterification conditions to form the following esterified polypeptide: CH3-A RG-LYS-ASP-VAL-TYR-OC2H5 EXAMPLES XXIX 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-A RG-LYS-ASP-VA L-TYR-R' which is substituted by the amino acids given in the following Table as indicated. Example No. R R' X H-GLN OH Xl H-GLU-GLN OH XII H-GLY-GLU-GLN OH XIII H-GLY-GLU-GLN VAL-OH XIV H-GLY-GLU-GLN VAL-GLN-OH XV H-GLY-GLU-GLN VAL-GLN-LEU-OH XVI H-GLY-GLU-GLN VAL-GLN-LEU-TYR-OH XVII H-GLN VAL-OH XVIII H-GLN VAL-GLN-OH XIX H-GLN VAL-GLN-LEU-OH EXAMPLE XX The polypeptide prepared as in Example I was acylated by reaction with acetyl chloride by known methods to prepare the following acylated derivative: CH3CO-ARG-LYS-ASP-VAL-TYR-OH EXAMPLE XXI The polypeptide prepared as in Example XX was also amidated by reaction with ammonia by known methods to prepare the following derivative: CH3CO-ARG-LYS-ASP-VAL-TYR-NH2 The polypeptide derivatives prepared in Examples VXXI retain the biological activity as described herein for the basic amino acid sequence. WHAT WE CLAIM IS:

1. A polypeptide having the capability of inducing the differentiation of Tlymphocytes but not of complement receptor (CR+) B lymphocytes, said polypeptide having the following sequence: R-ARG-LYS-ASP-VAL-TYR-R' wherein R and R' are terminal groups on said polypeptide which do not substantially affect the biological capability thereof, and the pharmaceutically acceptable salts, and wherein R and R' are independently selected from

R H OH C1-C7 alkyl NH2 C6-C12 aryl NHR, C7-C20 alkaryl N(R7)2 C7-C20 aralkyl OR7 C2--C7 alkanoyl C2-C7 alkenyl VAL-OH C2-C7 alkynyl GLN-OH N(R7)2 LEU-OH H-GLU TYR-OH H-GLY VAL-GLN-OH H-GLU-GLN VAL-LEU-OH H-GLY-GLN VAL-TYR-OH H-GLY-GLU GLN-LEU-OH H-GLY-GLU-GLN GLN-TYR-OH GLN-VA L-OH LEU-TYR-OH LEU-LEU-OH TYR-LEU-OH VAL-GLN-LEU-OH VAL-GLN-LEU-TYR-OH wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C6-C20 aryl, C7-C20 aralkyl, or C7-C20 alkaryl.

2. A polypeptide of the following sequence: R-ARG-LYS-ASP-VAL-TYR-R' wherein R is hydrogen, C1-C7 alkyl, C6C,2 aryl, or C2-C7 alkanoyl and R' is OH, NH2, NHR7, or N(R7)2, and the pharmaceutically acceptable salts.

3. A polypeptide of the following sequence: H-ARG-LYS-ASP-VAL-TYR-OH and the pharmaceutically acceptable salts.

4. A polypeptide according to Claim 1, wherein R is CH3CO and R' is OH.

5. A polypeptide according to Claim 1, wherein R is CH2 and R' is OH.

6. A polypeptide according to Claim 1, wherein R is H and R' is NH2.

7. A polypeptide according to Claim I, wherein R is H and R' is NH(CH3).

8. A polypeptide according to Claim 1, wherein R is H and R' is N(C2H5)2.

9. A polypeptide according to Claim 1, wherein R is CH3CO and R' is NH2.

10. A polypeptide according to Claim 1, wherein R is H and R' is OCH3.

11. A polypeptide according to Claim 1, wherein R is H and R' is OC2Hs.

12. A polypeptide according to Claim 1, wherein R is phenyl and R' is OH.

13. A polypeptide according to Claim 1, wherein R is benzyl and R' is OH.

14. A polypeptide according to Claim 1, wherein R is para-, meta- or orthotolyl and R' is OH.

15. A polypeptide according to Claim 1, wherein R is H-GLN and R' is OH.

16. A polypeptide according to Claim 1, wherein R is H-GLU and R' is OH.

17. A polypeptide according to Claim 1, wherein R is H-GLY and R' is OH.

18. A polypeptide according to Claim 1, wherein R is H and R' is VAL-OH.

19. A polypeptide according to Claim 1, wherein R is H and R' is GLN-OH.

20. A polypeptide according to Claim 1, wherein R is H and R' is LEU-OH.

21. A polypeptide according to Claim 1, wherein R is H and R' is TYR-OH.

22. A polypeptide according to Claim 1, wherein R is H and R' is VAL-GLN OH.

23. A polypeptide according to Claim 1, wherein R is H-GLN and R' is VAL OH.

24. A polypeptide according to Claim 1, wherein R is H-GLU-GLN and R' is VAL-GLN-OH.

25. A polypeptide of the following sequence:

R1 R2OR2 R4 a-R6-ARG-LYS-ASP-VAL-TYR-Resin wherein R1. R2, R2, R4 and R5 are protecting groups and wherein R1, R2, R2 and R4 are protecting groups on reactive side chains and wherein the R5 protecting group on the alpha amino acid is removable under conditions which do not affect the R1, R2, R, and R4 groups, and the Resin is an insoluble polymer having a functional group capable of being attached to the L-tyrosyl radical by a covalent bond.

26. An intermediate polypeptide according to Claim 25, wherein R1 is tosyl. R2 is 2-chlorobenzyloxycarbonyl, R3 is benzyl, R4 is 2,6-dichlorobenzyl, and R6 is t-amyloxycarbonyl.

27. A therapeutic composition comprising a polypeptide of Claim 1 in a pharmaceutically acceptable carrier.

28. A therapeutic composition according to Claim 27, wherein the amount of the polypeptide in the carrier ranges from 0.1 to 10 mg/kg of body weight.

29. A method for manufacture of the polypeptide of Claim 1 when R is H and R' is OH which comprises esterifying L-tyrosine, protected on its amino and hydroxvl groups, to an insoluble resin polymer by covalent bonding; removing the a-amino protecting group from the L-tyrosyl-resin, reacting with an a-amino protected Lvaline to couple L-valine to the L-tyrosyl-resin; removing the amino protecting group from the L-valine moiety, reacting with an amino protected L-aspartic acid to couple L-aspartic acid to the L-valyl-L-tyrosyl-resin; removing the amino protecting group from the L-aspartic acid moiety, reacting with an amino protected L-lysine to couple L-lysine to the L-aspartyl-L-valyl-L-tyrosyl-resin; removing the amino protecting group from the L-lysine moiety, reacting with an amino protected L-arginine to couple L-arginine to L-lysyl-L-aspartyl-L-valyl-Ltyrosyl-resin; and removing all protecting groups from the peptide.

30. A method according to Claim 29, wherein reactive side chains on the reacting amino acids are protected during the reaction.

31. A method according to Claim 29, wherein the resin polymer is selected from the group consisting of cellulose, poly(vinyl alcohol), poly(methylmethacrylate), sulfonated polystyrene, and a chloromethylated copolymer of styrene and divinylbenzene.