CS133091A3 - Human and rat thymopoietin - Google Patents

Description

-1-

Human and rat thymopoietin

SUMMARY OF THE INVENTION

The present invention relates generally to an isolated protein of purified form, from human or rat thymus, or produced by chemical synthesis or recombinant: genetic engineering methods, suitable for diagnostic purposes.

Background Art

Thymopoietin (hereinafter referred to as " TP ") is thyme polypeptide hormone with pleiotropic action, prothymocyte, mature T, the nicotinic acetylcholine receptors and hypophyseal corticotropins. Considerable attention has been paid to these substances and many articles and patents have been published relating to substances present in bovine thymus and isolated therefrom as synthetic peptides with properties similar to those of naturally occurring substances, e.g. : G.Goldstein, Nature ,. 247: 11-14 (London) (1974) RSBasch et al., Proc.Nati.Acad.Sci., USA, 71: 1474-1478 / 1974/1 MPScheid et al., J.Exp.Metf 147: 1727-1743 (1978) MPScheid et al., Science, 190: 1211-1213 / 1975 / GE

Ranjes et al., J.Exp.Med., 156: 1057-1064 (1982) J K. Venetasubramanian et al., Proc. Nat.Acad.Sci., USA, 83: 3171-3174 (1986); M. G. Malaise et al., In "Immunoregulatory UCLASymposium" ion Molecular and Cellular Biology, published by G. Goldstein et al., Liss, New York (1986); and G. H. Sunshine et al.; J. Immunol., 120: 1594-1599 (1978). (i, T. * _ «τ" ΊΤ - η Λ

The complete sequence of fifty amino-acid Tp (hereinafter referred to as HbTP / y work) was determined by T.Audhya et al., Biochemistry, 20: 6195-6200 (1981), and biological activity was attributed to the peptapeptide ARG-LYS-ASP- VAL-TYR, called thymopentin / Sal, referred to as "TP-5" / -z- and described by G. Goldstein et al., Science 204: 1309-1310 (1979) and Weksler et al., J.Exp.Med. , 148: 996-1006, as well as in MS patents Nos. 4190646 and 4261886. Furthermore, U.S. Patents 5,416,173, 4,420,424 and 4,629,723 disclose various peptides having thymopoietin-like activity, cited herein for completeness. extensive theses in the context of bTH, as well as in the development of synthetic peptides with the effects of b.TP but still, human thymopoietin has not been isolated from human thymus, nor has these proteins been fully sequenced so far from rat thymus tissue.completely sequenced or synthesized.

SUMMARY OF THE INVENTION The present invention relates to human thymo-poietin protein (referred to as MhTP * /) substantially free of human non-thymopoietin natural substances, i.e., free of such substances occurring together with the protein in its environment in the human. thymu. In particular, the invention relates to hTP having the following sequence of amino acid residues: GIT-LEU-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR-LYS-GLN-LYS-LEU-LYS-SER-GLU -LEU-VAL-ALA-GLY-VAL-THR = TEU-PRO-ALA-GLY-GLU-MET-ARG-LYS-ASP-VAL-TYR-VAL-GLU-LEU-TYR-LEU-GLN-HIS -LEU-THR-ALA-LEU-HIS. Further, the present invention relates to a protein cryhothymopoietin (referred to as "rTP"), essentially a non-mismopoietin natural sequence having the following amino acid residue sequence: -3-GLY-MET-PO-LYS-GLU-VAL_PRO -ALA-VAL-LEU-THR-LYS-GLN-LYS-LEU-LYS-SER-GLU-LEU-VAL-ALA-LYS-GLY-VAL-ALA-LEU-PRO-ALA-: GLY-GLU-GLN- ARG-LYS-ASP-VAL-TYR-VAL-ASP-ILE-TYR-PRO-GLN-i-HIS-LEU-THR-ILE-LEU-HIS-LYS-Aflg. ':

These proteins can be isolated from human or cryogenic thymus. Alternatively, these proteins can be synthesized chemically or prepared by recombination or chain polymerization / PCR techniques. <Γ Ϊ

Yet another aspect of the present invention relates to a method for diagnosing or detecting an amount of thymopoietin in a circulatory system that aggregates a step utilizing the above-described proteins. Another aspect of the invention encompasses DNA sequences comprising expression sequences encoding DNA. human or rat TP polypeptide. * r

The invention also provides recombinant molecules. . ? DNA containing DNA sequences encoding hTP or rTP in operative association with an expression control sequence. The present invention

they also provide trash cells with "these" molecules for the purpose of producing recombinant hTP or rTP. These? the molecules contain vectors or plasmids that exist extra-chromosomally in the host cells, or molecules that do; they integrate thymopoietin genes into host cell chromosomes. í í · v · i ·

Vectors and transformed cells of the invention are used. in another aspect of the invention, a novel process for producing recombinant human or rat thymopoietin. With this | in the method, the cell line is transformed with a DNA sequence encoding the respective TP. The human thymopoietin (hTP) or rat thymopoietin (rTP) DNA sequence is operably linked to the appropriate expression control sequence in the cell. The transformed cells are then cultured by conventional means in a suitable medium. This claimed process can use multivalent cells as host cells for polypeptide expression? Current preferred cell lines are mammalian cell lines and bacterial cells.

Yet another aspect of the present invention are anti-hTP or rTP antibodies. These antibodies are generated by using hTP or rTP, or fragments thereof, as immunogenic agents in conventional techniques for producing antibodies or polyclonal antisera. These anti-hTP or anti-rTP antibodies can be used as diagnostic or therapeutic agents.

In addition, the invention relates to therapeutic compositions of substances comprising a therapeutically effective amount of hTP as defined above, which may contain, among other ingredients, a pharmaceutically acceptable carrier. Specifically, the therapeutic effective amount of the polypeptide may be at least about 1 to 1000 µg / kg body weight.

Other aspects and advantages of the present invention will become apparent from the following detailed description. "

Brief description of drawings

Figure 1 graphically depicts the chromatographic analysis of human thymus extracts on the Sepha- dex G-75 molecular sieve when measuring immunoreactive RTP probTP. Lower molecular weight peak fractions were further purified to provide hTP.

Figure 2 graphically depicts the result of separation of CN3rdigest hTP after dissolution in 0.1% orthophosphoric acid with a Cg / Vydec 218TP54 reverse phase column at 46 mm x 25 cm / -5- at a flow rate of 2 ml / min. The peaks are labeled I, IIa III. The gradient line shows the presence of an organic solvent.

Figure 3 graphically depicts repetitive yields of phenylthiohydantoin-derivatized amino acids during Edman degradation of hTP using 46 cycles.

Description of the invention

The present invention provides two novel polypeptides, human thymopoietin and rat thymopoietin, produced in a form substantially free of contamination with other mammalian proteinaceous substances. Such polypeptides or modifications thereof are useful for diagnostic purposes for detecting abnormal levels of thymopoietin or as therapeutic agents for the treatment of various immunological diseases. In this specification, the amino acid residues of the peptides and certain substances used in their preparation are referred to as convenient abbreviations.

In fact, this isolation included briefly, the extraction of tissue in an aqueous solvent, ultrafillation for approximate distribution, hyorography, and the like. Phobic Chromatography and Reverse Phase HPLC Approximately 50% of the immunoreactive substance in each extract was present in a higher molecular weight form, which probably represents the form of a biologically inactive precursor (DFSteiner et al., Proc. Nat.Acad.Sci.USA, 57: 473). Xato was separated on a Sephadex G-75 molecular network Despite the simplification of the isolation procedure, the hTP yield was very low (0.42%), the product purification required -207 s-steps (Table 1), Large losses of hTP were formed by HPLC. The purity of hTP was evaluated by polyacrylamide gel electrophoresis, HPLC and amino acid sequence determination (see FIG. 3).

The procedures used to determine the amino acid sequence are also described in detail in Example 2 below. Residues 1-46 were determined by automated Edman degradation using a gas phase sequencer and residues 1-11 and 32-46 were verified by Edman degradation of two CNBr fragment fragments. This provided an independent confirmation of an important active region, residues 32-36. The C-terminal sequence of 44-48 is revealed by carboxypeptidase cleavage by time * * ·

These procedures gave the following amino acid sequence in hTP: 1 10GIY-LEV-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR — LYS-GLW-LYS- in 20 LEU-LYS-rSER-GLU -LEU — VAL-ALA-ASIT-GLY-VAL-THR-LEUvPRO-ALA-. -v. * · -. 30 40 GLY-GLU-MET-ARG-LYS-ASP-VAL-TYR-VAL-GLU-LEU — TYR-LEU-GLN— 48 HIS-LEU-THR-ALA-LEU-HIS. '.

Rat thymopoietin was isolated from rat thymus, and the sec-4 was dedicated as described below in the Examples. 5-7. Rat TP was further characterized by its substantially similar immunogenicity. human TP. These procedures gave the following sequence of fifty amino acids in rTP, 1 10GLY-MET-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR-LYS-GLN-LYS-. - 2θ LEU-LYS-SER-GLU-LEU-VAL-ALA-LYS-GLY-VAL-ALA-LEU-PRO-ALA-30. 40 GLY-GLU-GLN-ARG-EYS-ASP-VAL-TYR-VAL-ASP-ILE-TYR-PRO-GLN-48 HIS-LEU-THR-ILE-LEU-HIS-LYS-ARG. The peptides of the invention, in addition to their isolation of human or rat thymus, can also be produced by recombinant techniques, P.CR procedures and chemical syntheses. - <characterization, of sequenced hTP. rTP; .by; bTP antibodies are used. Previously, cross-reactivity of anti-bTP antibodies has been used to detect and isolate a closely similar bSP polypeptide from bovine spleen (T.Audhya et al., Biochemistry, 20: 6195-6200 / 1981). Similarly, antibodies to hTPP showed cross-reactivity to hTP ark monitoring hTP isolation from human thymus extracts using bTP radioimmunoassay (RIA) described in the above reference, which is incorporated herein by reference for completeness. Compared to the polypeptides of the known sequence, it has been found that hTP has a similar amino acid sequence as human soils (referred to as MhSP * ') as disclosed in co-pending patent application No. 53186 filed May 22, 1987, incorporated herein by reference. hSP has the sequence: 1 10GLY-LEU-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR-LYS-GLN-LYS-x. 20, ASN. LEU-LYS-SEFI-GLU-LEU-VAL-ALA-ASN / VAL-THR-LEU-PRO-ALA- 30 x 40

GLY-GLU-MET-ARG-LYS-ALA-VAL-TYR-VAL-GLU-LEU-TYR-LEU-GLN x x 48 SER-LEU-THR-ALA-GLU-HIS. -8-

Uak hTP so. hSPs are linear polypeptides of 48 amino acids. However, these polypeptides differ in four residues at amino acid positions 23, 34, 43 and 47, respectively, with an asterisk (*). _. . ......

HTP was also found to be similar to bTP and bSP / bovine spinach. bTP has the following amino acid sequence: 1 '' 1 1 10PRO-GLU-PHE-LEU-GLU-ASP-PRO-SER-VAL-LEU-THR-LYS-GLU-LYS-20 LEU-LYS-SER-GLU-LEU- VAL-ALA-ASN-ASN-VAL-THR-LEU-PRO-ALA-30 40 GLY-GLU-GLŇ-ARG-LYS-ASP-VAL-TYR-VAL-GLU-LEU-TYR-LEU-GLN-49 SER -LEU-THR-ALA-LUU-LYS-ARG. bSP has an amino acid sequence: 1 10PRO-GLU-PHE-LEU-GLU-ASP-PRO-SER-VAL-LEU-THR-LYS-GLU-LYS-20 "LEU-LYS-SER-GLU-LEU-VAL-.ALA -ASN-ASN-VAL-THR -LEU-PRO-ALA- 30 40 GLY-GLU-GLN-ARG — LYS-GLU-VAL-TYR-VAL-GLU-LEU-LEU-GLN-HIS- 49 LEU-THR- ALA-LEU-LYS-ARG Ten amino acid residues that are identical to hTP and hSP / amino acids at positions 1-4, 6, 8, 13, 31, 48 and 49 are at the same positions in the sequence In addition, hTPs differ from all of these peptides by the amino acid at position 23 · bTP and hTPs exhibit substantially the same active site / amino acid residue region 32-36), with the region being different however, only bTP and hTP do not exhibit such similar immunogenicity as hTP and rTP. -9- .... These comparisons confirm that the hTP 'amino acid sequence was indeed determined and indicated that during gene conversion sustaining parallel sequence development TP and SPu polypeptides of each species.

Other important gene systems that utilize gene conversion include: immunoglobulins (PHSchreier et al., Proc. Natl. Acad. Sci., USA, 78: 4495-4499 / 1981 // genes and geny major histocompatibility complex / M.Steinmetz et al. ., Science, 222: 727-733 / 1983 // * Keeping parallel development of the TP and SP regions outside the active site region (residues 32-36) leads to the conclusion that. these C- and N-terminal molecules must also have important functions; with similar claims. TP and SP maintained for each species. BTP residues 32-36 are known to represent an active site that corresponds to synthetic. TP / amino acid residues 32-36 / pentapeptide; , TP-5, having the biological activity of TP in animals and humans (G. Goldstein et al., Science, 20.4: 1309-1310 / 1979) and T.Audhya et al., Proc. Natl. Séi., USA, 81: 2847-2849 (1984). This study confirmed that active sites and rTPs (residues 32-36) were positive for bTP, so synthetic TP-5 / Arg-Lys-Asp-Val-Tyr / i also represented an active site in humans. The structure of rat thymopoietin is more similar to human than bovine thymopoietin, where it differs from human in positions 2,22, 25, 31, 38, 39, 41 and 46 and from beef in 1,2,3,4,6 positions. , 8,13, 22, 23, 25, 38, -39. 41, 43 and 46. Except for the alteration of Gln (rat) to human (human) and human (rat) to Ala (human), which require a change of the two bases in these codons, all changes of the acid peptide to the human represent a single base change in the codons. For bovine thymopoietin, 40% of the sequence changes from rat thymopoietin to this concept are two-base change, and the remaining 60% correspond to single base changes. ^ rez and hTP exist, about 83 &amp; homology between hTPq hTP -ip nl ari 77 T-, and πιοκί rTP with hTP λρ n / a hnmnl ησΐ p -10- about. 69% · The known active site (Arg-Lys-Asp-Val-Tyr) is all three proteins of the same (Goldstein). Science, 204: 1309-1310 (1979); T.Audhya et al., Biochem., 20: 6195-6200 (1981) and T.Audhuya et al., Proc.Nati.Acad.Sci., 84: 3545-3549 (1987). Homology between hTP and rTP provides substantially similar immunogenicity to these peptides. Thus, rTP has close cross-reactivity with anti-hTP antibodies and anti-rTP antibodies are cross-reactive against hTP. This implies that antibodies to rTP can replace antibodies to hTP a or rTP can be replaced by hTP in diagnostic assays and certain therapeutic assays, if desired. -, ζ £.

The present invention also encompasses DNA sequences coding for the hTP and rTP amino acid sequences. Variations of allele DNA sequences encoding hTP or rTP, as well as analogs or derivatives thereof, are also encompassed by this invention. The present invention includes these DNA sequences, without association with DNA sequences encoding other primate proteins and encoding the expression of hTP or rTP polypeptide. These DNA sequences include sequences that hybridize under stringent hybridization conditions. (see T.Maniatis et al., olecular cloning (A LaboratoryManual), Cold Spring Harbor Laboratory (1982), pp. 387-389). An example of one such stringent hybridization is the hybridization at 4XSC at 65 ° C followed by washing in 0.1XSSC. 65 ° C for 30 minutes. An alternative example of stringent hybridization conditions is 50% form, 4XSSC at 42 ° C. DNA 'sequences that hybridize to the hTP or rTP coding sequence under mild hybridization conditions and which express the expression of hTP or rTP peptides having hTP or rTPbiological properties also encode novel polypeptides. Examples of such mild hybridization conditions include 4XSSC at 50 ° C or hybridization of 30-40% formamide at 42 ° C. For example, a DNA sequence that shares distinct regions of homology, eg, glycosylation sites or disulfide bonds, hTP or rTP sequences, and encodes a protein having one or 4-11-multi-hTP or rTP biological properties, of course code-hTP or rTP polypeptide, when the DNA sequence does not hybridize strictly to the hTP or rTP sequence.

Similarly, DNA sequences that encode the hTP or rTP polypeptide but which differ in the codon sequence caused by the gegeneration of the genetic code or variation of the allele / naturally occurring base change in the species population, may or may not resulting in a change in amino acid (s) included in the present invention. Variations in the hTP or rTP DNA sequences, which are caused by point mutations or induced modifications to enhance the efficiency, of the half-life or production of the polypeptides thus encoded are also encompassed by the invention.

The present invention also provides alternative methods for the isolation of tissue, thymus, for the production of hTP and rTP. polypeptides.

One method of the invention involves culturing a suitable cell or cell line that has been transformed with a DNA sequence encoding the expression of hTP or .rTP polypeptides. or. its active fragment under the control of a known regulatory sequence. The regulatory sequences include promoter fragments, terminal "fragments" and other "suitable" sequences "that" direct "expression of the protein in a particular host cell.

Suitable cells or cell lines may be mammalian cells such as Chinese hamster ovary (CHO) or 3T3 cell cells. Selection of suitable mammalian host cells and methods for their transformation, culture, amplification, screening, and product manufacturing and purification are known in the art. See, eg, Gething and Sambrook, Nature, 29.3: 620-62-5 (1981), or alternatively Kaufman et al., Mol.Cell.Biol., 5/7 /: 1750-1759 / 1985 / or Howley and U.S. Patent No. 4,419,446. Other suitable cell lines include monkey COS-1 cell line and CV-1 cell line. In the present invention, bacterial cells may also be suitable as host cells, provided that the molecule produced retains activity in partially or wholly wound, state, or folded state, based on differences in glycosylation resulting from facial expression. -tor in mammalian and bacterial cells. For example, various E. coli strains are well known as host cells in biotechnology. Various strains of other bacteria, e.g. B.subtilis; can also be used in this process.

Also, many yeast cell strains will be available to those skilled in the art as host cells for the expression of polypeptides of the invention. In addition, insect cells can also be used as host cells in the process of the invention, if desired. See, for example, Tiller et al., Genetic Engineering, 8: 277-298 Alenum Press 1986 and references therein. The present invention also provides: recombinant molecular molecules, e.g., vectors and plasmids, for use in the ex-? new hTP and / or rTP. These molecules contain novel hTP or rTP DNA sequences that encode the hTP or rTP poly peptides of the invention. The vectors include truncated or altered hTP or rTP fragments, their allelic variants or modified sequences as described above, belong to such embodiments of the invention and are useful in the production of TP or rTP. The vector used in this procedure also contains selected regulatory sequences operably linked to the DNA encoding sequences of the invention and is capable of directing their expression and expression in selected host cells.

Alternatively, these peptides can be prepared by the following solid phase synthetic techniques originally described by Merrifiel, JACS, 85: 2149-2154 (1963). These processes are also disclosed in certain patents of the art which have been previously cited. Further procedures can be found, for example, in M.Bodanszky et al., "Peptide Synthesis" by John Wiley and Sons, Second Edition (1976), as well as in other works known to those skilled in the art. useful for thosynthesis and their abbreviations can be found in the aforementioned work as well as in JPWMcOmie, "Protective Groups in Organic Chemistry" / Plenum Press, New York. Conventional protecting groups used include tert-butoxycarbonyl (BOC), benzyl (BZL), tert-amyloxycarbonyl (AOC), tosyl (TOS), O-bromophenylmethoxycarbonyl (Br 2), and (2) 6 dichlofbenzyl (BZLC) and phenylmethoxycarbonyl (Z or Cbz).

More commonly, synthetic human thymopoietin is produced with a GLN .alpha.3 position, replacing MET in naturally occurring sequences. It has been found that the naturally occurring MET is subject to oxidation and makes the hTP unstable.

A still further process for preparing hTP and rTP polypeptides according to the invention leads through chain construction techniques (PGR). "PCR can be used to amplify and detect the desired nucleic acid from a mixture containing this sequence by others. hTP and rTP sequences from thymus or other tissue sources or human or rat lines by PCR Using this approach, they design two primers for each side of the sequence, hTP neborTP, one in the orientation and the other in the opposite sense. the primers are mixed with the biological sample or cell line to expand the appropriate extended products that act as templates for the synthesis of full length hTP or rTP nucleic acid sequences and for the detection of amplified sequences, which is described in detail in U.S. Patent Nos. 4,683,195 and 4,683,202. Since the secrets of hTP and rTP are known, the art The inventors can easily design appropriate appropriate to isolate and amplify large amounts of hTP / or rTP from biological sources by PCR. . The polypeptides of the invention or active fragments thereof can be used in the process for the development of monoclonal antibodies and / or polyclonal antisera. These antibodies regenerate using hTP or rTP, which, in essence, resemble impjt &amp; nocite, fragments thereof, or their modified or allelic versions, as antigens. By employing standard techniques for generating antibodies to those skilled in the art, e.g., by Kohler and Iilstein techniques, or by improved methods thereof, polyclonal, non-monoclonal antibodies that are suitable for diagnostic or therapeutic purposes can be produced.

Therefore, a further aspect of the invention includes diagnostic agents that comprise all or parts of hTP or rTP sequences. reagents, optionally in conjunction with detectable signals of various conventional types. can be used to diagnose disease states that exhibit either reduced or elevated TP levels. These disease states include, but are not limited to, myasthenia. gravis, diseases of immune deficiency, immunologically induced choroids such as arthritisrheumatoides, systemic lupus. erythematosus> and allergies, cancer, malnutrition and infections. One suitable assay using hTP and / or rTP is described in detail in Example 5 below. Other TPs are. other classes of assays may use either hTP or rTP polypeptides, fragments thereof, or antibodies developed through these polypeptides as described herein. The use of these diagnostic reagents to determine the levels may be appropriate to control the efficacy of the therapy: for each of the aforementioned conditions. -15-

Given the immunomodulatory properties of hTP and the present invention, it also encompasses the therapeutic use of those polypeptides in the treatment of humans and animals. These polypeptides are capable of inducing differentiation of lymphopoietic stem cells in hemopoietic tissues into thymus-derived cells7 / T cells / which are capable of engaging in the immune response of the organism. For this reason, these polypeptides are considered to have multiple therapeutic uses. * to its broadest application, these substances are suitable to pre-regulate the immune system of a subject, human or animal, in need of such regulation. The term &quot; regulate " as used herein means that the compounds cause the immune system to be adjusted from an abnormal, disease state of a normal, balanced state. First of all, said compounds have the ability to act in certain indicated functions of the thymus and therefore have an area of application in various functions of thymus and immunity. These compounds are generally considered to be useful in any area where cellular immunity is absent, particularly where there is an immune deficiency such as DiGeorg's syndrome and congenital absence of thymus.

The hTP and rTP of the invention can also be used to treat conditions with increased immunological activity. If there is an excess of antibody production due to T cell and B cell imbalance, said polypeptides can be used to treat this condition by stimulating T cell production. This suggests that these polypeptides are expected to be therapeutically useful in certain autoimmune diseases, including the generation of harmful antibodies such as jesystemic lupus erythematosus, arthritis rheumatoides and the like. -16-

In addition, these peptides are believed to contribute to the overall immunity of the organism by enhancing or contributing to the therapeutic stimulation of cellular immune and thus useful in the treatment of diseases involving chronic infections (such as fungal or mycoplasma). infection, tuberculosis, leprosy, acute and chronic and viral infections.

The present invention therefore also includes methods for regulating the immune system of a subject in need of such regulation, which comprises administering to the subject a therapeutically / immunoregulatory effective amount of one of the subject compounds as well as pharmaceutical compositions for practicing such procedures. The term " therapeutically effective amount " as used herein refers to an amount effective to treat conditions resulting from T cell deficiency or thymic deficiency, and the present invention provides a method of treating conditions off-selective or solitary , a thymic or T / deficient cell of a given system (human or animal), which includes a therapeutically effective amount of hTP to that subject.

For example, administration of one of said peptides to a patient suffering from DiGeorge's syndrome contributes to overcoming this deficiency. One skilled in the art of immunology can readily determine the appropriate dosage (preferably parenteral) and effective amount of one of said peptides to treat DiGeorge syndrome.

The present invention also provides a method of inducing lymphopoietic stem cells in a subject to develop characteristic lymphocyte lymphocytes comprising administering an effective amount of hTP inducing induction to a subject. The present invention further provides pharmaceutical compositions for practicing such procedures. In the preparation of the pharmaceutical compositions of the invention, hTP as an active ingredient is combined with a pharmaceutical carrier according to conventional pharmaceutical procedures for the manufacture of dosage forms. The carrier may be varied according to the administration form of the pharmaceutical form, e.g., sublingual, rectal, nasal, oral or parenteral. For the preparation of oral dosage forms, any suitable pharmaceutical media may be used: such as, for example, water, oils, alcohols, flavoring agents, preservatives. agents, dyes and the like in the case of oral liquid preparations / e.g. suspensions, elixirs, and solutions / or carriers such as starches, sugars, diluents, granulating additives, glidants, binders, disintegrating agents and the like in the case of oral solid preparations, e.g. Also, controlled release forms can be used to form powders, tablets and tablets. For reasons of ease of administration, tablets and boluses represent the most advantageous oral dosage form for which solids are used as pharmaceutical carriers. If desired, tablets may be impregnated. sugar or may be enterosolvent using standard procedures. In the preparation of parenteral formulations, the carrier is usually sterile water, although other agents may be included to aid solubility or preservation (for example). Injectable suspensions may also be prepared using suitable liquid carriers, suspending agents, and the like. hTP is generally active on parenteral administration above about 1 µg / kg body weight. For the treatment of DiGeorge's syndrome, the S-peptides can be administered parenterally from about 0.1. to 10 mg / kg body weight. In general, the same dose range can be used to treat other diseases or conditions that have already been reported, where immune deficiency is needed. Larger doses / e.g. and 10 to 100 mg / kg body weight / are suitable for suppressing excessive immune activity. 18- The following examples illustrate the isolation of hTP and process for the production of hTP and / or rTP by recombination. However, these examples do not limit the scope of the invention in any way. EXAMPLES Example 1

Isolation of human thymus hTP fresh human thymus derived from pediatric surgery and human surgical or autopsy spleen were cut and stored at -35 ° C. Separation agents and devices were obtained commercially: Sephadex (Pharmacia), hydroxy-apatite / Biogel HPHT 1 Bio-Rad / and preparative column with a high pore anion of HPLC / 2.5 x 25 cm, DE 500 , particle size 40-60 µm / Separation 1ndustries, Methanen, NJ). Purification of hTP was followed by the RIA method with bTP, which has cross-reactivity with hTP / G. Goldstein, J. Immunol., 117: 690-692 (1976). and P.J. Lisi et al., Glin.Chem.Acta, 107: 111-119 / 1980 //. Briefly, bTP was labeled by the method of Bolton-Hunter (A.E.Bolton et al., Biochem. J., 133: 529-539 / 1973) and the radiolabeled bTP was purified as described above. written by A.Audhya et al., Arch.Biochem.Biophys., 234: 167-177 (1984). The anti-bTP antibody (1: 10,000 dilution) was incubated with 125 I bTP / 10000 cpm / in the presence of a buffer of various peptide fractions in phosphate buffer for 8 hours at 22 ° C. Separation of bound and free bTP was performed with 24% polyethylene glycol at room temperature followed by centrifugation at 2000 rpm for 15 minutes. The resulting bTP was determined on (LKB S spectrum).

A batch of 714 g of human thymus was extracted with cold 100 mM ammonium bicarbonate / 25 wt%, 50 µg of 2-mercaptoethanol, 175 µg of phenylmethylsulfonylfluoride and 375-119 µg of ethylenediaminetetraacetic acid (EDTA) in 1 ml / homogeneous reducing in Waring Mixer (Dynamics, New Hartford, CT). Centrifugation at 14,000 x g at 4 ° C for 10 minutes, the supematant was filtered through a canvas and stored at 4 ° C after the addition of 0.1% thiomersal and 0.1% sodium azide. The soluble extract was subjected to ultrafiltration at 4 ° C using an Amicon Diaflo x M 100 membrane (cut-off molecular limit 100,000) and concentrated to Diaflo UM2 / cut-off molecular limit JT 1000 / with a volume reduction of 1/10 to 1/50.

Methateate from UM2 filtration, containing proteins relative to 1000-1000,000 molecular weight, was chromatographed on Sephadex G-75 granular, · · particle size 40020 / µm / at room temperature on a 5 x 150 cm column in 50 mM sodium bicarbonate the immunoreactive fractions were lyophilized. Two major peaks were found, containing immuno-reactive substances (cf. FIG. Higher molecular weight proteins have not been characterized. Further molecular weight peaking agents (M 3000, -8000) were further chromatographed on a hydroxyapatite column using Bio-Gelu HPHT / column 2.5 x 60 cm, 5 µM phosphate buffer sodium pH 6.8 /, where the samples were applied in the same "buffer."

Elution was carried out with 30 ml of the same buffer and then with 35 ml of 50 mM sodium phosphate buffer. The immunoreactive fractions were collected, concentrated on an Amicon Diaflo UM2 membrane, desalted on a 0.6 .mu.m 30 cm Sephadex G-25 (fine) column in 10 mM sodium bicarbonate (pH 8.0) and lyophilized. hTP was processed by HPLC using a preparative column of DE 500 with an anion exchanger of 2.5 x 25 cm. The lithium solvent gradient was initiated with 20 microliters of acetate buffer (pH 6) and terminated with 80% dilution of the same buffer with 500 ml of sodium acetate. The immunoreactive fractions were collected, dialyzed on a Spectrapor -o / cut-off molecular limit - M 1000 and lyophilized. Further chromatography was performed on a Vydac / 218TP54 reverse phase column using a linear gradient initially with 50 nM formic ammonium (pH 6.5) and 60% dilution with acetonitrile at the end. Eluted HPLC fractions were partially concentrated, lyophilized. and redissolved in 500 µl of 50 mM bicarbonate ammonium (pH 8). Volumes of 25 µl were evaluated by double RIA and the immunoreactive fractions were collected. and lyophilized. The yields from the hTP purification steps are shown in the table for immunoreactive total protein yield protein mg r% number of purification 1st lag cleaning stage of ultrafiltration 68000 309.5 0.45 1 100 gel filtration 2950G-75 76.9 2.6 6 25 * hydrophob. chromatog. 181.2 32.6 18 40 11 prep.EPLC 3.4 2.8 82 182 0.9 Chrome with rev. Phase 1.4 1.3. 93 207 0.42 Lower Molecular Weight Example 2

Sequence analysis hTP

Amino acid analysis was performed on a Liquimat III amino acid analyzer after acid hydrolysis for 2 hours -21-

with L y. at 100 ° C in an environment of 5.7 M HCl containing 0.5% 2-mer-2-tert-butanol (D. H. Speckman et al., Anal. Chem., 30: 1190),

1206/1958. ; 'M

The maltated hTP byp was prepared by the addition of maleic anhydride in 1,4-dioxane (100 µl) in a 20-fold excess relative to the free amino groups of the polypeptides (500 µg / t which were dissolved in 100 mM sodium tartrate / pH 9.3) /. The maleic hydride was added gradually over a 4 hour interval at which the pH was. maintained at 9.3 with 6M NaOH. £

This maleinated protein was then desalted on Bio-Gel P-2 ϊ in 100 mM ammonium bicarbonate (pH 8.2) and then lyophilized.

HTP trypsin maleate digestion was performed in 0.2 M N-ethylmorpholine acetate buffer (pH 8.1) at 37 ° C for 6 hours. Diphenyl pancreatic trypine (diphenylcarbamyl chloride) N &lt; 1 &gt; -r benzoyl-L-arginone-ethyl ester units per mg protein, type Sigma: XI / in the final enzyme / substrate weight ratio 1 was added. During the digestion, the pH was maintained at 8.5 and then reduced to 2.0 by addition of 10 mM HCl to quench the reaction. HTP (200 pmol) was digested with CKBr. at 150 yul. 70% formic acid for 23 hours, used in the dark at room temperature. 200-fold excess of CNBr relative to the methionine content of the sample. The volume was then reduced to 60 µl under nitrogen and applied directly to a glass filter of a gas phase sequencer (M.W.Hunkapiller et al., Methods Enzymol., 91: 399-413 / 1983). <T 1 * f · -22-

Trypsin digested peptides and digested CNBrbs were purified as follows. The tryptic peptides were dissolved in 0.1% otophosphoric acid (pH 2.2) and then centrifuged at 15600 x g for 5 minutes. Supernatant was introduced in a reverse phase column for HPLC / Vydac .218TP54) 46 mm x 25 cm, 5 µm particle size, The Separation Group, CA CA, LDC Spectrum Monitor II Module and Cl — 10 Integrator in conjunction with LKB Redirac 2112 /. A linear gradient elution was used initially using 0.1% phosphoric acid and 80% acetonitrile at the end. A high purity acetonitrile grade Burdich and Jackson / Liuskegon, MI was used, which was distilled in glass. Phosphoric acid (1%) was filtered through a filter; Milipore HA 0.45 µm (Milliore). All solvents were degassed under vacuum for 20 minutes.

Automated sequence analyzes of intact hTP and tryptic and cyanogenic hTP fragments were performed by sequencing in a gas phase sequencer in a gas phase model 47ŮA Applied Biosystems using Polybrene as a carrier and a standard single-coupling sing-cleavage program The resulting phenylthiohydantoin-derivatized amino acids were identified by HPLC using Hewlett Packard 10843 High Pressure Liquid Chromatography (PHSchlesinger, Methods Enzymol., 91: 494-502 / 1983). C-terminal analysis of hTP (150 [mu] g / each) was performed with yeast carboxypeptidase Y (2 nmol) in 100 mM sodium acetate / pH 6.0 at 37 [deg.] C. Aliquots were removed from the digest at various intervals and introduced into the test tubes. containing / µl of glacial acetic acid and thiobutyl was lyophilized. The sample was then dissolved in 0.2M sodium acetate (pH 2.2) and applied to an amino acid analyzer. At the same time, enzyme and peptide blank samples were processed. As a result of the above procedures, the yield was 1.3 mg of immunoreactive. hTP (0.4% yield / Table 1) from about 1.4 kg of freshly frozen human thymus. This product had one main band and several minor bands at isoelectric gel focusing. The isoelectric point of the main lanes was 6.1 + 0.2, Table 2 below shows the amino acid composition of hTP in molar ratios.

Table 2

Amino acids found average + SK cysteine acid 0 ND aspartic acid 2.29 +0.12 threonine 3 3.11 + 0.23 serine. 1 0.81 + 0.19 glutamic acid 6 5.55 + 0.22 proline 3 2.80 + 0.30 glycine 3 2.28 +0.27 alanine 4. 4.25 + 0.10 valine 6. 6,23 + 0,30 methionine ': 1' // 0,87 + 0,13 isoleucine 0 0,10 +0,11 leuein 9 8,72 + 0,36 tyrosine 2 1,90 +0,12 phenylalanine 0 1 0.16 + 0.11 lysine 5 4.92 + 0.31 histidine 2 1.76 + 0.12 arginine 1 1.07 + 0.13 ND - not detected

Diameter of two determinations / 24 and 76 hours / ,. The value for serine was increased by 10% and for threonine by 5% to compensate for acid destruction. Hydrolysis was performed with constant boiling HCl at 110 ° C in vacuo. Cleavage by hTP with cyanogen bromide yielded 3 differentiated fractions (see Fig. 2) and the amino acid sequence results for each fraction are summarized in Table 3.

Table 3

N-terminal degradation method of CNNBr cleavage of native TPp1

Peak II C-terminal carboxypeptidase method

peak III

peak IV

peak V C-terminal carboxypeptidase method identified by residues 1-46 1-11 32-46 44- 4837-47 no sequence obtained 18-30 45-48

The hTP structure was determined by automated N-terminal sequencing of 240 'pmol hTP at 46 cycles of Edman degradation, which yielded a repetitive yield (mean yield per cycle / 53.4%) and identified all of the 46 N-terminal residues (see FIG. 3). . The release of the C-terminal amino acids of the intact hTP by carboxypeptidase tabulka (Table 4) gave a non-equivocal tetrapetide sequence with two amino acids overlapping (residues 45 and 46) with the N-terminal sequence, the partial hTP sequence was determined by determining the CNBr fragment I / fig structure Which includes residues 32-40 and which contains an active site. In addition, the amino acid composition of intact hTP / ta [alpha] 2 corresponds to all amino acids found by sequence analysis. Table 4 below shows the C-terminal free amino acid release time of the free amino acids by carboxypeptide Y.-25-

Table 4 amino acids yield, nmol

Amino Acid LOOs 120s 150s 200s 250s 300s Thr 75 210 263 Ala - - 88 258 276 288 Leu 148 275 283 418 503 His 289 310 300. 308 299 Example 4

Isolation of TPP from thyme. Frozen rat, thymus was thawed on ice, condensed and excess fat removed. Thymus (74 g) was homogenized in ice-cold 0.5 M ammonium bicarbonate (25% feedstock, pH 8.3) containing 50 ng of 2-mercaptoethanol, 175 µg of phenylmethylsulfonyl fluoride and 375 µg of disodium salts of ethylenediaminetetraacetic acid in buffer. Insoluble matter was removed by centrifugation at 17,000 x g for 60 minutes. The supernatant was filtered through a screen and bacteriostatic, thiomersal aazide sodium was added, each at a final concentration of 0.1%. ·.

The extract containing the solutes was treated in a 350 ml Amicon stirred cell using a 100-100 / 100,000 mole membrane. weight dividing value /, the filtrate was concentrated / 740ml. to 70 ml / on a YM-2/2000 mol.-weight separator membrane and the reteritate was subjected to column chromatography on Sephadex G-50 / particle size 20-80 µm / using a 2.5 x 100 cm glass column room temperature in a solution of 0.05M ammonium bicarbonate buffer (pH 8.3). Samples of a volume of 20 ml in each sub-step (flow rate -26- in 40 ml / h) of the thymopoietin-containing fraction were loaded. were detected by radioimmunoassay (GwGoldstein, J. Immunol., 117: 690-692 / 1976) and lyophilized. . t ‘

The teyl protein content determined by Coomassie Bluepro Protein Assay (Pierce, Rochford, IL) according to the manufacturer's instructions and lyophilized protein was processed on a high-resolution molecular network as described below.

Sephacryl HR-100 (Pharmacia, Pistaway, NJ) was charged to a Pharmacia XK 16/70 column coupled with an effective, rapid, liquid chromatography (FPLC) system, and 0.02 M ammonium bicarbonate was charged into the loading and elution buffer. (pH 7.0) at a column bed volume of about 130ml. Lyophilized protein (2.0-3.0 mg / was resuspended). 1.3 ml of buffer and loaded onto a column with 1.0 ml of a syringe, eluting the column at a flow rate of 1.5 ml / min. The gel filtration column was monitored. UV uptake by LKB-Unicord-S-II coupled to a two-channel recorder, collecting 1.5 ml fractions.

Corresponding fractions from several chromatographic runs. were collected and protein content determined by O.D. at 280 nm. Then, these fractions were lyophilized in silicone glass jars and their immunoreactivity determined as described below. Example 5

RTH immunoreactivity

Rat thymopoietin isolated by the procedure of Example 4 was determined using a rabbit anti-human thymopoietin anti-substance. This antiserum was incubated with various concentrations of -27- soluble protein isolated on Sephackryl ColumnHR-100 for 2 hours at 370µl on a rocker. This mixture was added to rat thymopoietin-coated polyvinyl chloride microtiter plates according to previously described procedures (T.Audhya et al., Bc. J. Immunol., 31: 199-204 / 1990) and incubated at 37 ° C for 2 h. After washing the plate with 0.15 M phosphate buffer pH 7.2 containing 0.05% Tween 20 (PBS-Tween 20), goat-anti-rabbit horseradish peroxidase / HRPO / Jackson Labs was added. , West Grove, PA, and the plate was incubated at 37 ° C for 1 h. After three successive washes with PBS-Tween 20, a HgO 2 -containing substrate-chromogenic system was added. 5 * -tetramethylbenzidine (TMB) (SigmaChemical St.Louis, MO) was allowed to react colorless at room temperature and in the dark for 30 minutes. density at 450 nm, in enzymatic immunoassay; (ELISA) using rat thymopoietin-coated plates; anti-human thymoprotein antibodies, the purified rat and human thymopoitin gave a parallel displacement curve, demonstrating an iminologic similarity, whereby the thymusbibi-thioblastin showed a %10.4% reactivity compared to human thymopoietin. and the difference from the above-mentioned bovine thymopoietin did not yield a parallel seal-out curve and showed less than 0.1% cross-reactivity with human thymopoietin in this immunoassay. Example 6

Polyacrylamide Gel Electrophoresis (PAGE) and WesternBlot Analysis; The protein sample obtained in Example 4 above was further characterized by treating 10 µl of this sample with 0.25 µl of 10% sodium dodecyl sulfate (SES) and 1 µL-28-bromophenol blue and electrophoresis on a PharmaciaPhast System using 20% homogeneous polyacrylamide gel. Prior to gel electrophoresis, 6-piece filter paper and one 0.2 micron nitrocellulose paper were wetted in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). Transfer. -sandwich; was assembled as follows: 3 pieces of filter paper, nitrocellulose paper, gel and 3 pieces of filter paper. The protein was then transferred to the PhastTransfer system under 6 min / gel and 25 mA / gel conditions. The membrane was then blocked overnight with 3% bovine serum albumin (SigmaChemical Co., St. Louis, M0) in PBS-Tween 20, then washed three times with PBS-Tween 20 and allowed to react with HRPO-conjugated monoclonal antibody to human muthymopoietin at The membrane was then washed three times in PBS. Tween 20 and incubated with substrate-chromogen / TMS membrane peroxidase, Kirkergaard Perry Labs, Gaithersberg, MD for 15 to 30 minutes. , washed with water and air dried. PAGS has been demonstrated. that rat and human thymopoietins are similar in molecular weight. Rat thymopoietin has demonstrated similar migration characteristics to that of the human thymopoietin in Western blotting. Example 7

Characterization of purified rat thymopoietin a. Amino acid composition: The amino acid complexity was determined to be four fold. 6NHC1 hydrolyzed polypeptide samples. at 110 ° C under vacuum for 24, 50, 71 and 94 hours and the values obtained were extrapolated to zero hydrolysis time. The analysis was carried out on an amino acid analyzer Qeckman6300 / ral Alto, CA by the method of Moore et al. 30: 1185-1190 (1958). -29-

b. Isoelectric focusing using PAGE

The isoelectric focusing method was used according to Finlayson and Chrambacha, Anal. Biochem., 40: 292-311 (1997), using a mixture with ampholine to provide a pH between 3.5 and 8. Isoelectric focusing method. frying thymopoietin was found. 6.8. c. Carboxypeptidase analysis of carboxy-terminal

Rat thymopoietin carboxy-terminal analysis was performed as described in T.Audhya et al., Proc.Nati.Acad.Sci., 84: 3545-3549 / 1987 /. The enzyme k.sub.2 substrate was 1: 200. The time-dependent analysis of carboxypeptothioase gave Arg, Lys and His in equimolar ratios. , d. HH-terminal sequence analysis

The fraction of purified protein (200 nmol) was fractionated by 4-20% SDS-PAGE at 50mA constant current. After electrophoresis, the proteins were transferred from the gel to a membrane of polyvinylidene difluoride / PVDF using a semi-dry electroblotter unit (Integrated Separation SystemS110201 / Hyde Park, MA) at 200V constant for 30 minutes. The membrane was then stained with Coomassie Blue and bleached. The PVDF membrane containing the blotted protein in the 5.5 kDa region was cut into a pieceyr2 x 4 mm and placed on top of the conditioned Biobrane filter as an inert carrier. Automated Edman. degradation was performed on an Applied Biosystems sequencer; Pulsed liquid 477A model (Applied Biosystems, Foster City, CA). Phenylhydantoin / PTH / amino acids have been identified on the Applied Biosystems PTH amino acid analyzer (Model ABI 120A). The eluent was detected at 269 nm. Automatic sequencing of 1000 pmol protein for 48 cycles from the N-terminus gave a repetitive yield of 93%. -30- Example 8

Expression, recombinant human or rat TP 1. To produce hTP or rTP with cDNA that encodes them? For example, such vectors are known in many types for mammalian, insect, yeast, fungal, and bacterial expression using standard molecular biology techniques. For example, the pUC series of vectors is commercially available. Other vectors for mammalian expression are described, eg, by pXM / Y.C.Yang et al., Cell, 47: 3-10 / 1986. The selected vector can be linearized with the appropriate endonuclease enzyme and sequentially ligated in an equimolar amount, separately, to a cDNA encoding hTP or rTP, which has been pre-modified by the addition of synthetic oligonucleotides that generate complementary ends to generate expression constructs. . These constructs can be expressed in different hosts with appropriate vectors

One of ordinary skill in the art can construct other mammalian expression vectors by, for example, inserting the hTP and rTP DNA sequences into the plasmid with the appropriate enzymes, using known genetic engineering methods, and other known vectors such as pUC18, which is commercially available from Amersham, Buckinghamshire , GB or Pharmacia, Uppsala, Sweden. a. Expression in mammalian cells

Vector for expression in mammalian nun. it is possible to synthesize positively well known art. The components of these vectors, for example, replicons, selected genes, enhancers, promoters and the like can be obtained from natural sources or synthesized by known techniques. See, eg, Kaufman et al., J. Kol. Biol. 159: 511-521 (1982) and Kaufman, Proc. Nat.Acad.Sci., USA, 52: 689-693 (1985). Examples of mammalian host lipids include, but are not limited to, primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid-31- cells, cell strains derived from in vitro primary tissue cultures as well as primary explants are also suitable. The proposed cells need not be genotypically deficient in the selected gene if this selection gene is dominant. Prostable integration of vectors. DNA and subsequent amplification of the integrated DNA vectors can be used in both CHO cell conventional methods. Alternatively, the DNA vector may comprise all or part of the bovine paulillus genome of the virus (Lusky et al., Cell, 36: 391-401 / 1984), which may be introduced into such cell lines as C 127 mouse cells as a stable episomal element . Other suitable cell lines include, but are not limited to, HeLa, monkey COS-1 cells, mouse cells. L-929, 3T3 line. derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. transforming hTP or rTP vectors into suitable host cells may result in expression of hTP or rTP polypeptides. Stable transformants are screened for expression, product expression by, standard immunological, biological, or enzymatic assays. The presence of DNA or mENA coding for hTP or rTP polypeptides can be detected by standard techniques such as Southern blocking or RNA 'blocking' transient expression of DNA encoding polypeptides within a few days after introduction of the DNA expression vector into suitable host cells such as COS-1 monkey cells. B, Bacterial Expression Systems The skilled artisan can process hTP or rTP encoding sequences by eliminating each mammalian regulator sequence, blocking the coding sequences, and introducing bacterial regulatory sequences generating bacterial vectors 32 for intracellular or extracellular expression of hTP neborTPs according to the invention by bacterial cells The DNA encoding hTP or rTP may be further modified so as to contain different codons to optimize bacterial expression as already known in the art. the maturehTP or rTP coding sequence is operably linked in frame to nucleotide sequences encoding the framework leader polypeptide, allowing bacterial expression, secretion and maturation of hTP or rTP polypeptides, also by methods known in the art. Expression of hTP or rTP in E. coli using such secretory systems may result in secretion of the active polypeptide.

Compounds expressed in some way from bacterial host cells can then be isolated, purified, and / or characterized in terms of physico-chemical, biochemical and / or clinical parameters by any known method. in c. Expression by insect or yeast cells

Similar methods can be used to construct an insect vector for the expression of hTP or rTP polypeptides in insect cells (see, eg, the procedures described in published European Patent Application No. 155476).

Similarly, yeast vectors are constructed using yeast regulator sequences to express cDNA encoding hTP or rTP in yeast cells to generate secreted extracellular active hTP or rTP / viz. the procedures described in published PCT application WO86 / 00639 and European patent application 123289 /. In each expression system described above, the resulting cell line can be further amplified with a suitable drug, the resulting cell lines can be recloned, and the level of expression can be. using the hTP or rTP assays described in this copy. -33- .ν ’. Example 9

ϊ, ': Diagnostic Assays Using hTP or rTP The following assay can be used to diagnose circulating-TP in tissue fluids of a selected patient for disease diagnosis or to monitor treatment efficacy, A.Radioimmunition - polyethylene glycol separation

To determine the level of Tp in a patient's cerebrospinal fluid sample, this sample is chromatographed on a Sephadex G-50 molecular network in 0.1 M ammonium bicarbonate at pH 8, and the Eractions between the secreted / Vo / and permeable / Vi values are collected and lyophilized and the lyophilisate is taken up in thyme-poietin buffer. The purpose of this treatment is to exclude molecules of molecular weight greater or less than thymopoietin from this assay. To 0.5 ml of hTP-antiserum. diluted in 5% bovine gamma-glottal in 0.01M. phosphate buffer - 0.15M NaCl. (BGG buffer) is added 0.2 mL of CSF sample of the selected patient and 0.3 µL of ITP or IrTP of the invention (50,000 cpm in SGG buffer). Incubation - - - - - - - carried out three times in 12 x 75 mm plastic tubes.

The tubes were vortexed and allowed to stand at room temperature for two hours. Thereafter, half a milliliter of cold 30% polyethylene glycol was added to each tube by vortex mixing and then tubes were centrifuged at 2000 rpm for 20 minutes in a cooled centrifuge. -bench activity using an automated gamma spectrometer.

The precipitated ω-hTP (in%) is calculated according to formula 1-a / bc / x 100, where α = cpm of the clot with the antibody, b = total, given cpm and c = cpm non-specifically precipitated with normal rabbit. lice sherry or. with an antibody and an excess of unlabelled thymmopoietin. (usually 10% of total cpm). In the standard curve, the binding inhibition of the thymopoietin binding was calculated as a percentage of the maximum thymopoietin binding with the antibody at 10 °. B. Radioimmunoassay - Double Antibody Separation The above procedure A is repeated except that 4M KCl BGG buffer is used and after 2 hours of incubation a total of 0.1 ml goat anti-rabbit antibody and 0.02 ml normal rabbit are added. antibodies wherein the components are mixed with a navor-mixer and the resulting mixture is mixed; Allow to stand at room temperature for 5 minutes and then centrifuge. Separate the supernatant and collect the precipitate for the radioactivity of Part A above. C. Radioimmunoassay-separation with activated charcoal coated with dextran

Dextran-coated activated charcoal is prepared by mixing the same volumes / i / 5 grams of Norit A charcoal in 100 ml phosphate buffer containing 2M KCl and bovine gamma globulin and / ii / 0.5 g dextran 110 in 100 ml of phosphate buffer containing 110 KCl and bovine garaaglobulin.

A total of 0 5 ml of dextran-coated charcoal was added at 4 ° C to the assay tube after 2 hours of incubation as described in Part B above and left to stand for 30 minutes at 4 ° C. The tubes are then centrifuged and the supernatants are aspirated. The pellets are then evaluated for radioactivity, which in this case represents the determined "unbound" thymopoietin. When determining the binding inhibition curve for unlabeled thymopietin using polyethylene glycol separation, the antigen-antibody complex exhibits a sensitivity to thymopoietic concentrations greater than 5 ng / ml. No major deviations are observed in control polypeptides including insulin, alga-bungarotoxin, ubiquitin, histone asynthetic tridecapeptide fragment of thymopoietin / residues 29-41 / at concentrations of 10-1000 ng / ml.

Binding inhibitory curves for unlabeled thymopoietin in these assays using double separation of antibodies and separation of the antigen-antibody complex. on dextran-activated charcoal exhibit thymopoietin sensitivity at concentrations below 0.1 ng thymopoietin per ml; Thus, the latter two methods are particularly suitable for measuring thymopoietin levels in CSP samples. The foregoing description includes many, modifications and variations of the invention, and it is believed that such solutions will be apparent to those skilled in the art. For example, the use of suitable vectors, vector components, and host cells for recombinant production of the polypeptides of the invention is within the skill of the art, and such modifications and changes in composition are obviously included within the scope of the invention.

Claims (4)

PATENT CLAIMS Rat Thymopietin substantially free of contamination with other proteinaceous material having a sequence of amino acid residues of GLY-MET-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR-LYS-GLN-LYS-LEU-LYS- SER-GLU-LEU-VAL-ALA-GLY-GLY-GLU-GLU-GLU-GLU-GLU-GLU-GLU-GLU-GLU-GLU-GLU-GLY-ASP-VAL.TYR-VAL-ASP-ILE-TYR-PRG- GLN-HIS-LEU-THR-ILE-LEU-HIS-LYS-ARG. A method for producing rat thymopofetin comprising culturing a cell line transformed with a DNA sequence encoding a rat thymopoietin or fragment in operable association with a control sequence thereof. -press 1 3. A cell transformed with the amino acid sequence of claim 1 in operative association with an expression control sequence. 4. A cell according to claim 3 which is a mammalian or bacterial cell. 5, 'A plasmid vector comprising the amino acid sequence of claim 1. -37-6'. A diagnostic agent comprising a mammalian thymopoietin amino acid sequence selected from the group consisting of GIY-MET-PRO-LYS-GLU-VAL-PRO-ALA-VAL-LEU-THR-LYS-GLN-LYS-LEU-LYS-SER-GLU- LEU-VAL-ALA-GLY-GLY-GLU-GLR-GLU-GLR-GLU-GLR-GLU-GLU-GLR LEU-THR- ILE-LEU-HIS-LY3-ARG. 7. A method for the thymopoietin level in a sample of a fluid of a patient's tissue, which utilizes all or part of the sequences of claim 6, optionally associated with radiolabeling, as a reagent. ii The method of claim 7, wherein the set. and. m, right? said tissue fluid is cerebrospinal fluid. . · ''. · Á '. i., a