Patent Publication Number: US-4258151-A

Title: Pentapeptide modified resin

Description:
The invention described herein was made in the course of work under a grant or award from the U.S. Department of Health, Education and Welfare. 
    
    
     CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a division of parent application Ser. No. 6,894, filed Jan. 26, 1979 now U.S. Pat. No. 4,190,647, dated Feb. 26, 1980, which is a continuation-in-part of Ser. No. 851,778, filed Nov. 15, 1977, and now abandoned, which is a continuation-in-part of Ser. No. 631,176, filed Nov. 11, 1975, and now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates generally to new polypeptides, to methods for preparation of the new polypeptides, and fields of use for the polypeptides. 
     2. Description of the Prior Art 
     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 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; Cell, 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 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 &#34;thymotoxin,&#34; was believed to cause myositis but it was further indicated that this polypeptide of approximately 7,000 molecular weight, had a strong net positive charge and was retained on CM-Sephadex at a pH of 8.0. 
     In the publication &#34;Nature,&#34; 247, 11, Jan. 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 U.S. Pat. No. 4,077,949, issued Mar. 7, 1978, from application Ser. No. 606,843, filed Aug. 22, 1975, which is a continuation-in-part of application Ser. No. 429,202, filed Dec. 28, 1973, and now abandoned. 
     In issued U.S. Pat. No. 4,002,602, dated Jan. 11, 1977, which is a continuation-in-part of application Ser. No. 449,686, filed Mar. 11, 1974, and now abandoned, there are disclosed long chain polypeptides described as 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 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. Pat. No. 4,002,740, dated Jan. 11, 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 named as thymopoietin in above-mentioned U.S. Pat. No. 4,077,949. 
     The present invention provides a synthesized five-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. Pat. No. 4,002,602, dated Jan. 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 polypeptides of this invention, as well as compositions and methods for use in biological actions. 
     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: ##STR1## as well as this peptide intermediate freed from the resin and other protecting groups, wherein X, Y and Z are as above, and R 1 , R 2 , and R 3  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 polypeptides 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: 
     
         X-Y-Z-GLN-LYS                                              I. 
    
     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: 
     
         R-NH-X-Y-Z-GLN-LYS-COR&#39;                                    II. 
    
     wherein X, Y and Z are as described above, and R and R&#39; 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&#39;) 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 pentapeptide as in some polypeptides. Therefore, it is considered that the scope of the present invention is inclusive of these pentapeptides 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 &#34;folding&#34; 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 &#34;fold&#34; in a certain way. In the present invention it has been discovered that the pentapeptide &#34;folds&#34; 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 &#34;folds&#34; 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 seventy-four amino acid peptide disclosed as Ubiquitin in U.S. Pat. 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&#39; in formula II can be any substituent that does not substantially affect the biological activity of the basic active sequence. Thus, for purposes of illustration R and R&#39; may be any of the following substituents: 
     
         ______________________________________                                    
        R                R&#39;                                               
______________________________________                                    
Hydrogen               OH                                                 
C.sub.1 -C.sub.7 alkyl NH.sub.2                                           
C.sub.5 -C.sub.12 aryl NHR.sub.7                                          
C.sub.6 -C.sub.20 alkaryl                                                 
                       N(R.sub.7).sub.2                                   
C.sub.6 -C.sub.20 aralkyl                                                 
                       OR.sub.7                                           
C.sub.1 -C.sub.7 alkanoyl                                                 
C.sub.2 -C.sub.7 alkenyl                                                  
C.sub.2 -C.sub.7 alkynyl                                                  
______________________________________                                    
 
    
     wherein R 7  is C 1  -C 7  alkyl, C 2  -C 7  alkenyl, C 2  -C 7  alkynyl, C 6  -C 20  aryl, C 6  -C 20  alkaryl, or C 6  -C 20  aralkyl. 
     As pointed out above however, R and R&#39; can also be amino acid groups or residues of polypeptide chains having 1 to 20 carbon atoms. The following are illustrative: 
     
         __________________________________________________________________________
R           R&#39;                                                            
__________________________________________________________________________
ASP       GLU                                                             
SER       SER                                                             
LEU       THR                                                             
SER--ASP  LEU                                                             
LEU--SER--ASP                                                             
          HIS                                                             
LEU--ASP  VAL                                                             
LEU--SER  ARG                                                             
          GLU--SER                                                        
          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: 
     
         R-HN-TYR-ASN-ILE-GLN-LYS-COR&#39;                              III. 
    
     wherein R is hydrogen, C 1  -C 7  alkyl, e.g. methyl, ethyl, C 5  -C 12  aryl, e.g. phenyl, or C 1  to C 7  alkanoyl, e.g. acetyl or propionyl and R&#39; is OH, NH 2 , NHR 7 , or N(R 7 ) 2 . The most preferred polypeptides are those wherein R is hydrogen and R&#39; 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, 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. The 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 U.S. Pat. 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 described in U.S. Pat. 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-1 (θ), 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. 
     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 through 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 application is in the treatment of DiGeorge Syndrome, a condition in which there is a congenial 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 B 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 becomes 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 in 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 polypeptides is its in vivo agility to restore cells with the characteristics of T cells and also its in vivo ability to restore cells with the characteristics of B cells. 
     A further important property of the peptides of this invention is that they are highly active at 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 polypeptides 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 basic pentapeptides of this invention were prepared using the concepts similar to those described by Merrifield as reported in Journal of American Chemical Society, 85, pp. 2149-2154, 1963. The synthesis involved is 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 was 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. 
     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, polymethylmethacrylate 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 protection. 
     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 α-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 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: ##STR2## 
     In the above sequence of reactions R 1  and R 2  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 α-amino group is removed to permit further reaction and α-R 3  is a protecting group of the α-amino group. Preferably, in the above intermediate pentapeptide resin, the term R 1  stands for a protective grouping such as 0-2, 6-dichlorobenzyl, R 2  stands for ε-2-chlorobenzyloxycarbonyl and R 3  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 R 1 , R 2 , and R 3  protecting groups thereon and the resin. The protecting groups are removed by conventional means, e.g., by treatment 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 may be cleaved from the substrate in HF to form C-terminal amides. Techniques for use of an N-methyl-benzhydrylamine resin are described by Monahan et al. in Biochemical and Biophysical Research Communications, 48, 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 alphaamino 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 substituents such as benzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to utilize tert-butyloxycarbonyl (t-BOC) or t-amyloxycarbonyl (AOC) for protecting the α-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since the BOC and AOC (t-amyloxycarbonyl) protecting groups are readily removed following such reaction and prior to the subsequent step (wherein such α-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 α-amino groups may be protected by reaction with any material which will protect the amino groups for the subsequent reaction(s) but which may later be removed under conditions which will not otherwise affect the molecule. Illustrative of such materials are organic carboxylic acid derivatives which will acylate the amino group. 
     In general, any of the amino groups can be protected by reaction with a compound containing a grouping of the formula: ##STR3## 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 isonicotinyloxycarbonyl, phthaloyl, para-tolylsulfonyl, formyl and the like. 
     In conducting the general process of the invention, the peptide is built by reaction of the free α-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 α-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: 
     
         H.sub.2 N-TYR-ASN-ILE-GLN-LYS-COOH 
    
     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-ε-2-chloro-benzyloxycarbonyl-L-lysine 
     Alpha-BOC-asparagine 
     Alpha-BOC-L-Isoleucine 
     Alpha-BOC-O-2,6-dichlorobenzyl-L-tyrosine 
     In these reagents, BOC is t-butyloxycarbonyl. &#34;Sequenal&#34; 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 0.75 mM of chloride per gram of resin. 
     In preparation of the polypeptide, 2 m moles of α-BOC-ε-2-chlorobenzyloxycarbonyl-L-lysine were esterified to 2 m moles of chloromethylated resin in absolute alcohol containing 1 mM triethylamine for 24 hours at 80° C. The resulting coupled amino acid resin was filtered, washed with absolute alcohol and dried. Thereafter, the other α-BOC amino acids were similarly coupled to the deprotected α-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 α-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 incomplete 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 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 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. 
     
         H.sub.2 N-TYR-ASN-ILE-GLN-LYS-COOH 
    
     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 BALB/c 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-1 congenic mice. 
     For the induction of in vitro of Thy-1 +  T cell or CR +  B cell differentiation, the induction of thymocyte differentiation from prothymocytes 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 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-1 -  and CR - ) because they have very few or no Thy-1 +  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-1 +  T cells in concentrations ranging from 10 ng to 1 μg/ml, and also induced the differentiation of CR +  B cells in concentrations of 10 ng to 1 μg/ml. 
     EXAMPLE III 
     The protected 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 tyrosine moiety. The resulting peptide is then acylated with acetic anhydride, followed by cleavage from the resin substrate and removal of all protective groups with HF. The following acylated derivative is thus produced. 
     
         CH.sub.3 CONH-TYR-ASN-ILE-GLN-LYS-COOH                     A. 
    
     EXAMPLE IV 
     To produce the amidated derivative of the pentapeptide 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 derivative is produced by cleaving the protected pentapeptide attached to the basic resin by reaction with hydrogen fluoride. The amidated pentapeptide has the following formula: 
     
         H.sub.2 N-TYR-ASN-ILE-GLN-LYS-CONH.sub.2                   B. 
    
     For identification, thin layer chromatography and electrophoresis were employed which provided the following data: 
     
         ______________________________________                                    
Thin Layer Chromatography:                                                
Sample: 30 μg                                                          
Silica Gel (Brinkman glass plate, 5 × 20 cm, 0.25 mm                
thickness)                                                                
R.sub.f.sup.1 : n-BuOH: Pyridine: HOAc: H.sub.2 O 30: 15: 3: 12           
R.sub.f.sup.2 : EtOAc: Pyridine: HOAc: H.sub.2 O  5: 5: 1: 3              
R.sub.f.sup.3 : EtOAc: n-BuOH: HOAc: H.sub.2 O 1: 1: 1: 1                 
Spray reagent: Pauly, Ninhydrin &amp; I.sub.2                                 
                            Electro-                                      
                            phoresis                                      
                 TLC        Peptide                                       
                                      moved to                            
Compounds:         R.sub.f.sup.1                                          
                          R.sub.f.sup.2                                   
                                 R.sub.f.sup.3                            
                                      cathode                             
______________________________________                                    
Ac--Tyr--Asn--Ile--Gln--Lys                                               
                   0.47   0.87   0.54 -1.35 cm                            
Tyr--Asn--Ile--Gln--Lys--NH.sub.2                                         
                   0.48   0.87   0.37 -6.60 cm                            
Electrophoresis:                                                          
Whatman 3 mm paper (11.5 cm × 56.5 cm)                              
Sample: 100 μg                                                         
pH 5.6, Pyridine-Acetate buffer solution                                  
1000 V, 1 hour                                                            
Spray reagent: Pauly &amp; Ninhydrin                                          
______________________________________                                    
 
    
     The acetylated pentapeptide of Example III, when utilized in a concentration of 1 μg/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 μg/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: 
     
         H.sub.2 N-TYR-ASN-ILE-GLN-LYS-COOCH.sub.3 
    
     EXAMPLE VI 
     The diethylamine derative 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 Example V is reacted with diethylamine in dimethylformamide solution to produce the following diamino-substituted amide. 
     
         H.sub.2 N-TYR-ASN-ILE-GLN-LYS-CON(C.sub.2 H.sub.5).sub.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 ε-2-chlorobenzyloxycarbonyl 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 protective group is then removed from the LYS alpha-amino acid to form the substituted polypeptide of the following formula: 
     
         C.sub.2 H.sub.5 -NH-TYR-ASN-ILE-GLN-LYS-COOH 
    
     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 substrate and the N-ethyl derivative is produced without cleaving 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: 
     
         C.sub.2 H.sub.5 NH-TYR-ASN-ILE-GLN-LYS-CONH.sub.2 
    
     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: 
     
         CH.sub.3 CONH-TYR-ASN-ILE-GLN-LYS-CONH.sub.2 
    
     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&#39; to provide the basic amino acid of the formula: 
     
         R-NH-TYR-ASN-ILE-GLN-LYS-COR&#39; 
    
     which is substituted by the amino acids given in the following table as indicated. 
     
         __________________________________________________________________________
EXAMPLE NO.                                                               
           R         R&#39;                                                   
__________________________________________________________________________
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                                
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 following pentapeptides are prepared: 
     Example XXII-H 2  N-ALA-ASN-ILE-GLN-LYS-COOH 
     Example XXIII-H 2  N-TYR-ALA-ILE-GLN-LYS-COOH 
     Example XXIV-H 2  N-TYR-ASN-ALA-GLN-LYS-COOH 
     To determine the pharmacological activity and characteristics of these pentapeptides, use was made of the induction of Th-1 (T-cell) antigen on chicken bone marrow for use as an assay of the peptides at a concentration of 1 μg/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 5×10 6  cells per milliliter with the peptide (0.1 μg/ml) in RPMI 1630 medium supplemented with 15 mM Hepes, 5 percent y-globulin-free fetal calf serum, deoxyribonuclease (14 to 18 unit/ml), heparin (5 unit/ml), penicillin (100 unit/ml), and streptomycin (100 μg/ml). Controls are incubated with BSA (1 μg/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-1 +  or Th-1 +  cells in each layer are calculated as a cytotoxicity index, 100 (a-b)/a, where a and b are the percentages of viable cells in the complement control and test preparation, respectively. The percentage of cells induced is obtained by subtracting the mean values in the control incubations without inducing agents (usually 1 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-1 (T-cell) antigen or chicken bone marrow 
     
         ______________________________________                                    
Example No.    % Induction                                                
______________________________________                                    
XXII           12                                                         
XXIII          15                                                         
XXIV           21                                                         
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     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.