MXPA97003549A - Stable dietilentriaminopentaacetic acid, jointed by the end of n: compositions of protein and met - Google Patents

Abstract

The present invention relates to dietarytriaminepentaacetic acid (DTPA): the compositions of the protein wherein the DTPA chelating agent is conjugated specifically located at the N-terminus of the protein, thereby providing a homogeneous and well-defined product capable of forming the complexes with a variety of metal radionuclides. The compositions of the present invention can be produced in large quantities and retain total bioactivity in vivo, both with and without the chelated metal radionuclide. The compositions can be used in potential in the diagnosis, the detailed production of the images, and / or in the treatment of leukemia and related diseases.

Description

STABLE DTPA UNITED BY EXTREME N: COMPOSITIONS OF PROTEIN AND METHOD FIELD OF THE INVENTION The present invention relates broadly to the field of protein modification, and, more specifically to diethyl ether-pyridine acid (DTPA): the composition is of the protein wherein the DTPA chelating agent is conjugated at the location specifically to the termination of N of the protein, thus the proportion of a homogeneous and well defined product able to form complexes with a variety of metallic radionuclides. In another aspect, the invention relates to the methods of conjugating DTPA to the stimulation factor of the granulocyte colony (G-CSF) or to the inter-eucin-2 (IL-2), thus the proportion of a useful procedure of radio-classification or labeling of proteins and related proteins that include cytokines, while maintaining the structural and functional integrity of the protein.

REF. 24631 BACKGROUND OF THE INVENTION The radioactive labeling or labeling of proteins and other biological compounds is commonly achieved by means of iodine treatment. Proteins can be classified or marked successfully with radiosotopes of iodine by a number of methods; Reogoeczi, E., Plasma Proteins Classified or Marked with Iodine l-_- 53 '(CRC Press, Boca Raton, Fia 1982), and the antibodies so classified or labeled are used in radioinuclear mutation studies in which the location of the tumor is determined by the detailed production of the external images; Keenan et al., J. Nucí. Med., 2_6_ 531 (1985). However, in the course of these investigations and others involving the use of iodine radioisotopes, certain limitations to the use of detailed production procedures of radioiodine images become apparent (eg, the characteristics of detailed production of the poor images of many of the radioisotopes of iodine, the classification or labeling procedures involved, and their high degree of instability of classification or in vivo labeling). In addition, the most common methods of iodine treatment involve oxidation conditions in the reaction mixtures that can modify other sensitive groups and cause alterations in the structure of the protein, and possible biological inactivation.

To avoid the difficulties encountered when trying to treat these proteins and other compounds with iodine, alternative methods are used. One of these methods is the "bifunctional chelate" method, in which the strong chelating groups are covalently bound to the proteins so that the chelate bound to the protein can then complex with a variety of metal radionuclides; Meares & Goodwin, J. Prot. Chem., 3_, 215-228, (1984), the paramagnetic metal ions; Lauffer & Brady, Ma g n. Re s or n. Imag. , 3_, 11-16, (1985); Ogan et al., Invest. Radlol. , 2_2_, 665-671, (1987), and fluorescent metals; Muk ala et al., Anal. Biochem. , 116, 319-325, (1989).

The most commonly used reagents for the covalent modification of proteins with a chelating agent is the cyclic dianhydride of DTPA. The cyclic dianhydride of DTPA generally forms stronger chelates than the analogs of ethylenetriaminetetraacetic acid (EDTA); Perrin et al., Organic League ds, (Organic Ligands) IUPAC Chemical Data Series No. 22, (New York, Pergamon Press 1982), and involves less complicated synthesis procedures than those involved when using EDTA analogues. In addition, the cyclic dianhydride of DTPA is stable indefinitely at room temperature, thus providing for greater control under the coupling conditions. Hnatowich and McGann, Int. J. Rad. Appl. Instrum. , [B] 14, 563-568 (1987). The coupling of DTPA to proteins usually develops at pH > 7.0, wherein the dianhydride first reacts with the free amino groups (e.g., available lysine residues) to form the amide bonds; Hnatowich et al., Scienc, 220, 613-615, (1983a).

A first interest in achieving these covalent modifications is that there may be many possible locations in each protein where the chelators may be linked. Existing current methods provide for non-selective ligation in any reactive group, either localized within the protein, such as a lysine side group, or at the N-terminus. This results in a heterogeneous population. For example, the reaction of DTPA dianhydride with insulin produces a complex mixture of several products, including the crosslinked protein and the acylated tyrosine residues; Maisano et al., Bioconj. Chem., 3_, 212-217 (1992), while the reaction of albumin with DTPA dianhydride produces the protein molecules with multiple linked chelating groups; Lauffer & Brady, Magn. Reson. Imag. , 3, 11-16, (1985).

The number of DTPA groups conjugated to the protein is often given as an average number, when the sample preparations are heterogeneous, each having the protein with either the chelating groups more and less than the average number; Hnatowich and McGann, Int. J. Rad. Appl. He instructed , [B], 14, 563-568 (1987). It is well known that proteins can be degraded by the covalent binding of the chelating groups, with the degree of degradation increasing with the increasing substitution; Sakahara et al., J. Nucí. Med., 2_6_, 750, (1985). These protein molecules that contain several chelating groups are less likely to retain their original biological properties; Meares and 'Goodwin, Jour. of Prot. Chem., 3_, 215-228 (1984). From a producers' point of view, regulatory regulatory approval for the salts of these heterogeneous therapeutic proteins may have additional complexities.

The in vivo properties of the proteins marked with the chelate were reviewed; Meares et al., Adv. Chem., 198, 369-387 (1982). The most general observations are that in vivo stability depends critically on the chemical nature of the chelation conjugate with the protein, that the proteins classified or labeled most clearly have the greatest biological half-life, and that the retention of activity becomes more probable by the procedures (eg classification or specific marking) that minimizing the classification or marking of the residues involved in the active location (s). For example, horse serum albumin (HSA) was conjugated with the chelating agent, classified or labeled with 111 I n, and when injected in vivo it was quickly cleared by the liver (when compared to the results following the classification). labeling of 125I in the HSA treated with iodine); Leung and Meares, Biochem. Biophys. Res. Commun. , 7J5_, 149-155 (1977). The HSA conjugated with the chelate, in at least the population with the chelating groups of greater number and so represent a large percentage of the radioactivity followed, can be recognized in vivo as the foreign protein; Meares and Goodwin, Jour. of Prot. Chem., 3_, 215-228 (1984). The advantage of avoiding the random and numerical distribution of the products through the classification or marking in a specific way of a simple (non-essential) location in the protein is evident.

The covalent coupling of DTPA to proteins using DTPA dianhydride was described by several investigators. For example, Khaw et al. , Science, 209, 295, (1980) DTPA coupled to immunoglobulin G (IgG) fragments active against myosin and investigated the location of protein classified or labeled in canine myocardial infarcts. Using the same method, Scheinberg et al., Science, 215, 1511, (1982) prepared the monoclonal antibody classified or labeled specific for cells e r i t r o 1 e u c a mi s in the mouse. Although these methods and others provide the coupled proteins, they are invariably characterized by complicated synthesis and by low coupling efficiencies. Hnatowich et al., Science, 220, 613-615, (1983a).

U.S. Patent No. 4,479, 930. { Hnatowich) describes compositions comprising a dicyclic dianhydride coupled to an amine, and chelated with a radioisotope metal cation. The compositions are reported to be stable i n v i v o. Methods of preparing the compositions are also described. It is reported that the initial and final pH of the coupling reaction mixture is pH 7.0 in all examples, and that the coupling efficiency (defined as the percentage of the anhydride molecules that are linked covalently to the polypeptide or the protein) is high when the molar ratios of the anhydride to the antibody are maintained at 1: 1, but the pH values decrease above or below neutrality. It is not taught as to the distribution of the DTPA portion in the proteins or polypeptides of the different reaction products.

Nothing can be extracted from the literature concerning the preparation of DT PA: conjugated protein that is in convenient form over those previously described due to the fact that conjugation is the specific location at the N-terminus of the protein, thus producing a well-defined, homogeneous composition. The compositions can be produced in large quantities and fully retain the bioactive activity, even with or without the chelated metal radionuclide. The synthesis described in the present invention is a simple one-step reaction wherein a simple located reagent is created, providing a useful method for the classification or labeling of the proteins. The conjugate of DT PA: p r o t a n of the present invention may have potential use in the diagnosis, detailed production of the images and / or treatment of leukemia and related diseases.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the homogeneous preparations in substantial form of the proteins chemically modified by the end of N, and to the methods thereof. Unexpectedly, the conjugation of the DTPA chelating agent is located specifically to the N-terminus of the protein, thus providing a more homogeneous and well-defined product as compared to other chelating agents: protein compositions. Also unexpectedly, the preferred DTPA: G-CSF conjugate can chelate a number of metallic radionuclides producing a radiolabeled or radiolabelled product, while maintaining the structural and functional integrity of the protein, and this method is broadly applicable to other (or analogous) proteins, as well as to G-CSF.

In one aspect, the present invention relates to a homogeneous preparation in substantial form of DTPA: G-CSF (or analogous thereof) and related methods. A working example below demonstrates that the DTPA chelating agent is specifically conjugated at the N-terminus of the rhG-CSF, and that such a composition is capable of forming the complexes with a variety of radionuclides. metallic Since the conjugation is specific to the N-terminus of the G-CSF molecule, the resulting product is a more homogeneous and well-defined product than previously described.

The present invention also relates to a method for the preparation of a classified or labeled protein, the method comprising: (a) the reaction of a chelating agent with the protein at an acidic pH sufficiently to selectively activate the a-amino group in the amino terminus of the protein; (b) the preparation of conjugated protein of the unconjugated protein; (c) the addition of a metal cation to the conjugate; and (d) obtaining the protein classified or labeled. This method is described below for rhG-CSF and IL-2, and this provides for additional aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows the effects of the initial pH and the molar ratio of D T PA: p r o t e n a in the coupling of rhG-CSF. The analysis of the SDS-PAGE of the following examples is done: line 1- MW markers; lanes or lanes 2-4, DTPA: rhG-CSF at 5: 1, 50: 1 and 500: 1, respectively, at the initial pH of 6.0; lanes or lanes 5-7, DTPA: r G-CSF at 5: 1, 50: 1 and 500: 1, respectively, at the initial pH of 7.0; lanes or tracks 8-10, DTPA: rhG-CSF in 5: 1, 50: 1, and 500: 1, respectively, at the initial pH of 8.0; lane or lane 11, rhG-CSF at pH of 6.0 lane or lane 12, rhG-CSF at pH 8.0.

FIGURE 2 shows the HPLC elution diagrams with the size exclusion for the rhG-CSF starting material (line 1), the DTPA conjugation reaction mixture: rhG-CSF before passing through a column of G50 rotation (line 2), and the reaction mixture of the DTPA conjugation: rhG-CSF after passing through a G50 rotation column (line 3). Elution is monitored for absorbance at 280 nm.

FIGURE 3 shows the elution diagrams of the FPLC with the exchange of the preparative cation for the starting material rhG-CSF (faded line) and the reaction mixture of the DTPA: rhG-CSF (solid line). Elution is monitored for absorbance at 280 nm.

FIGURE 4 shows the HPLC analysis by analytical cation exchange of the rhG-CSF (lines 2 and 3) and the conjugate of DTPA: rhG-CSF (lines 1 and 4) preincubated with 111In. Elution is monitored for absorbance at 220 nm (lines 3 and 4) and for radioactivity (lines 1 and 2, inverted).

FIGURE 5 shows the HPLC analysis by the exchange of the analytical cation of the rhG-CSF (line 1), the DTPA conjugate: rhG-CSF (line 2), and the DTPA conjugate: rhG-CSF treated with excess InCl3 (line 3). The elution is monitored for absorbance at 220 nm. EDTA (1 mM) is added to Shock Absorber A.

FIGURE 6 shows the analysis of the silica gel TLC plate of the following samples: lane or lane 1, 111In 0.1 nmol (indium with a trace of 111In); lane or lane 2, 111In 10 nmol aggregates to DTPA 20 nmol; lane or lane 3,, 1, In 10 nmol incubated with rhG-CSF 2 nmol, followed by the addition of DTPA 20 nmol; and lane or lane 4, 111In 10 nmol incubated with the DTPA conjugate: rhG-CSF 2 nmol, followed by the addition of DTPA 20 nmol. For lanes or lanes 2-4, aliquots containing 111In 0.1 nmol are taken from the mixtures and loaded onto the plate.

FIGURE 7 shows the MALDI-MS spectrum of the DTPA conjugate: rhG-CSF. The spectrum shows the protonated species multiplied (1, 2, 3 and 4 bound protons).

FIGURE 8 shows the ion sprayed mass spectrum of the DTPA conjugate: rhG-CSF with the chelated indium. The conjugate is pre-incubated with the saturation of InCl3 (I n: with water, 10: 1, mol / mol) before analysis.

FIGURE 9 shows the ion atomized mass spectrum of the rhG-CSF.

FIGURE 10 shows the peptide mapping of the DTPA conjugate: rhG-CSF. Peptide fragments generated from the conjugate of DTPA-rhG-CSF (solid line) and rhG-CSF (vanished line) by proteolysis are reduced, alkylated, and then redissolved by HPLC with inverted phase. Elution is monitored for absorbance at 215 nm. The arrow indicates the elution of the N-terminus peptide from the digested sample of rhG-CSF.

FIGURE 11 shows an isoelectric focusing gel (pH 3-10) containing the following samples: lane or lane 1, rhG-CSF; lane or lane 2, conjugate of DTPA: rhG-CSF preincubated with I n C 13 in excess (In: conjugate, 10: 1 mol / mol); track or track 3 conjugate of DTPA: rhG-CSF; and path or track 4, markers of the isoelectric point.

FIGURE 12 shows the spectrum of circular dichroism (CD) of the conjugate of DTPA: rhG-CSF without () and with () chelated indium, and unmodified rhG-CSF () and DTPA (_ • _ •) • Samples (0.078 mg / ml protein, and 4.07 μM DTPA) are analyzed at 10 ° C in 20 mM sodium acetate, pH 5.4. The sample of the unmodified rhG-CSF and the DTPA sample: rhG-CSF (without indium) are identical.

FIGURE 13 shows the effects of DTPA conjugation of the activity i n vi v o of the rhG-CSF. Activity (WBC count) is measured after subcutaneous injection of hamsters (cotton rat). The dose of rhG-CSF was 100 μg / kg. The bars represent - Il ¬ The standard deviation (n 8-10 for the protein samples, and n 5-6 for the baseline).

FIGURE 14 shows the HPLC analysis by analytical cation exchange of IL-2 (lines 2 and 3) and the DTPA: IL-2 conjugate (lines 1 and 4) preincubated with 111In. Elution was monitored for absorbance at 220 nm (lines 3 and 4) and for radioactivity (lines 1 and 2, inverted).

FIGURE 15 shows the HPLC analysis by analytical cation exchange of IL-2 (line 1), DTPA conjugate: IL-2 (line 2), and DTPA: IL-2 conjugate treated with excess InCl3 (line 3) . The elution is monitored for absorbance at 220 nm. EDTA (1 mM) is added to Shock Absorber A.

FIGURE 16 shows the analysis of the silica gel TLC plate of the following samples the track trail 111 In 0.1 nmol Indian with a trace of 111 In); path or track 2, 111 In 10 nmol added to DTPA 20 nmol; lane or lane 3, 111In 10 nmol incubated with IL-2 2 nmol, followed by the addition of DTPA 20 nmol; and lane or lane 4, 111In 10 nmol incubated with the DTPA conjugate: IL-2 2 nmol, followed by the addition of DTPA 20 nmol. For lanes or lanes 2-4, aliquots containing 111In 0.1 nmol were taken from the mixtures and loaded onto the plate.

FIGURE 17 shows the peptide mapping of the DTPA: IL-2 conjugate. Peptide fragments generated from the conjugate of DTPA-IL-2 (solid line) and IL-2 (vanished line) by proteolysis are reduced, alkylated and then re-dissolved by HPLC with inverted phase. Elution is monitored for absorbance at 215 nm. The arrow indicates the elution of the peptide with N-terminus from the digested IL-2 sample.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The conjugate of DT PA: p r o t e n t of the present invention is described in more detail in the discussion that follows and is illustrated by the examples provided below. The examples show the different aspects of the invention and include the results of the biological activity analysis of the different DTPA: protein conjugates. Surprisingly, using the methods of the present invention, a location of the simple reagent is created such that the conjugation of the resulting DTPA is in specific location at the N terminus of the protein, yielding a well defined and homogeneous composition capable of complexing with a variety of metallic radionuclides, while maintaining the structural and functional integrity of the protein.

A variety of cytokines and related proteins are contemplated for use in the practice of the present invention. Exemplary proteins contemplated include the various topo-ethical factors such as the aforementioned G-CSF, GM-CSF, M-CSF, interferons (alpha, beta, and gamma), interleukins (1-14), erythropoietin (EPO), fibroblast growth factor, stem cell factor (SCF), growth factor and megalovirus development (MGDF), platelet derived growth factor (PDGF), and tumor growth factor (alpha, beta).

The stimulation factor of the granulocyte colony (G-CSF) is a glycoprotein that induces the differentiation of the cells of the hemapoietic precursor to neutrophils and stimulates the activity of mature neutrophils. The G-CSF (rhG-CSF), recombinant human expressed in E. c or l i, contains 175 amino acids, has a molecular weight of 18,798 Da, and is iologically active. Currently, Filgrastima, a G-CSF, recombinant, is available for therapeutic use.

The structure of G-CSF under different conditions has been studied extensively; Lu et al., J. B i or l. Ch em. Vol. 267, 8770-8777 (1992), and the three-dimensional structure of rhG-CSF was determined recently by means of x-ray crystallography. The G-CSF is a member of a class of growth factors that share a common structure, decorative motif of a pack of four a-spirals with two large crossover connections; Hill et al. , P.N. A. S. USA, Vol. 9_0_, 5167-5171 (1993). This family includes the growth hormone of GM-CSF, interleukin-2, i n t e r 1 eu c i na-4, and interferon β. The extension of the secondary structure is sensitive to the pH of the solvent, where the protein acquires a high degree of content of 1 to 1 h a 1 i c or i a 1 at the acidic pH; Lu et al. , Arch. Biochem. Biophys. , 286, 81-92 (1989).

In general, the G-CSF useful in the practice of this invention may be an isolated form of mammalian organisms or, alternatively, a product of synthetic chemical procedures or of the expression of the prokaryotic or eukaryotic host of the exogenous chains of DNA obtained by genomic cloning or cDNA or by synthesis DNA Suitable prokaryotic hosts include the different bacteria (e.g., E. coli); suitable eukaryotic hosts include yeast (e.g., S. cerevisiae) and mammalian cells (e.g., ovarian cells of Chinese hamster, chango cells). Depending on the host employed, the expression product of G-CSF can be glycosylated with mammals or other eukaryotic carbohydrates, or they can not be glycosylated. The expression product of G-CSF can also include an amino acid residue of the initial methionine (in position -1). The present invention contemplates the use of any and all forms of G-CSF, despite the fact that recombinant G-CSF, especially derived E. coli, is preferred, among other things, for superior commercial utility.

Certain analogous G-CSFs have been reported to be biologically functional, and these can be chemically modified. Analog G-CSFs are reported in U.S. Patent No. 4,810,643. Examples of the other analogous G-CSFs reported to have biological activity are those set forth in Australian Patent A-76380/91, European Patent O 459 630, European Patent O 272 703, European Patent O 473 268 and European Patent O 335 423, although no representations are made with respect to the activity of each analog described as reported. See also Australian Patent A-10948/92, North American PCT No. 94/00913 and European Patent 0 243 153.

In general, the G-CSFs and analogs thereof useful in the present invention can be investigated by practicing the procedures for chemical modification as provided herein and the analysis of the resulting product for the desired biological characteristics, such as the assessment of the biological activity provided here. Of course, if so desired when treating non-human mammals, the non-human G-CSF's, ecominants, such as murine, bovine, canine recombinants, etc. can be used. See the PCT International No. 9105798 and the PCT International No. 8910932, for example.

The first one, a glycoprotein with a molecular weight of approximately 15,000 daltons, is a member of the group called lymphokines that intervenes in the immune responses in the body. This protein is produced by activated T cells and is known to have several activities in vivo. For example, IL-2 is reported to increase thymocyte mitogenesis, induce T-cell reactivity, regulate gamma-interferon, and enhance the recovery of immune function of lymphocytes in the selected immunocytochemical states. This has potential application in the search for the treatment of neoplastic diseases and immunodeficiency and has been used in therapies for the treatment of cancer.

IL-2 useful in the practice of this invention may be an isolated form of mammalian organisms or, alternatively, and especially in an analogue of IL-2, a product of synthetic chemical procedures or expression of the exogenous DNA chains of the prokaryotic or eukaryotic host obtained by genomic cloning or cDNA or by the synthesis of DNA. Suitable p r o c a r i t t i c t o rs include various bacteria (e.g., E. C or l i); suitable eukaryotic hosts including yeast (e.g., S. c e r e v i s e a) and mammalian cells (e.g., Chinese hamster ovary cells, chango cells). Depending on the host employed, the IL-2 expression product can be glycosylated with the mammalian or other eukaryotic carbohydrates, or these may not be g 1 u c or s i 1 a d s. The IL-2 manifestation product may also include an amino acid residue of the initial methionine (in position -1). The present invention contemplates the use of any and all forms of IL-2 and its analogs, although recombinant IL-2 and analogues, especially E. The derivative is preferred, for, among other things, the higher commercial utility.

Methods for the preparation of IL-2 are known by isolation and purification of the form that is present naturally or by genetically engineered means. See, for example, US Patent Nos. 4,778,879; 4, 908,434; and 4, 925, 919 (Mertelsman et al.); U.S. Patent No. 4,490,289 (Stein); U.S. Patent No. 4,738,927 (Taniguchi et al.); U.S. Patent No. 4,569,790 (Koths et al.); U.S. Patent No. 4,518,584 (Mark et al.); U.S. Patent No. 4, 902,502 (Nitecki et al.) and European Patent No. 0136489 (Souza et al.).

The IL-2 receptor (IL-2R) is overexpressed constitutively in several ema to 1 malignancies, including adult T cell leukemia (Uchiyama, et al., 1985), hairy cell leukemia ( Trentin, et al., 1992), chronic lymphocyte leukemia (Rosolen, et al., 1989), Hodgkin's disease (Strauchen and Breakstone, 1987), and non-Hodgkin's lymphoma (Grant, et al., 1986). Lymphocytes involved in several autoimmune diseases, including rheumatoid arthritis (Lemm and Warnatz, 1986) and allograft rejection (Waldmann, 1989) also overexpress IL-2R. This receptor has therefore been actively pursued as a target for cytotoxic therapy. Toxins from the recombinant fusion were produced where the cell ligation domain of the pseudomonas P exotoxin (L oberbo um-G a 1 ski, et al., 1988a) or the ligation domain of the toxin receptor of diphtheria (Williams, et al., 1987) was replaced with IL-2. These fusion proteins are cytotoxic in specific form to the cells that manifest the higher affinity of IL-2R (Loberboum-Galski, et al., 1988b, Williams, et al., 1990). A recently described chimeric exotoxin / IL-4 P pseudomonas protein may also prove useful for the treatment of autoimmune diseases, allograft rejections and many malignancies in which the cells manifest elevated levels of the IL receptor. 4 (Puri, et al., 1994). A human G-CSF fusion protein of the diphtheria-related toxin has also recently been made which may have utility in the study and treatment of leukemia (Chadwick et al., 1993). The conjugation of DTPA to IL-2, IL-4, as well as rhG-CSF may also have potential use in diagnosis, detailed production of the images and / or treatment of leukemia and related diseases. Chemometric radiometals for cytotoxic therapy may include 212Bi, 211At, and 90Y. In fact, the antibodies for the IL-2R chain, and that carries the 12Bi and 90Y radioisotopes by means of the chelates if 1 is conjugated, are being examined by several investigators (Junghans, et al., 1993; Parenteau, et al. ., 1992; Kozak, et al., 1990) for cytotoxicity towards the T cell lines to 1 orreactives, and for potential radiotherapy.

The DTPA useful in the conjugations of the present invention is the technical grade DTPA dianhydride.

In a preferred embodiment involving E. c or l i derived from rhG-CSF, the conjugation of DTPA: rhG-CSF which is present at an initial pH of 6.0, and a molar ratio of DTPA: rhG-CSF 50: 1.

In a preferred embodiment involving E. c or l i derived from IL-2, the conjugation of DTPA: IL-2 occurs at an initial pH of 6.0, and a molar ratio of DTPA: IL-2 50: 1.

Although the investigations are described and illustrated with respect to DTPA conjugates: specific protein and treatment methods, it will be apparent to a skilled in the art that a variety of related conjugates, and treatment methods may exist without departing from the scope of the invention.

The following examples will illustrate in more detail the different aspects of the present invention.

EXAMPLE 1 The DTPA used for conjugation is initially the dianhydride form, and there is therefore the potential for unwanted side reactions such as protein: protein crosslinking; Hnatowich et al., J. Imm u n o. Me t h o d s, 65, 147-157, (1983b). Reaction conditions such as the initial pH and the molar ratio of the DTPA dianhydride: rhG-CSF were investigated in order to minimize the formation of such products. The rhG-CSF was produced using recombinant DNA technology where the E cells. c or l i t r a n s f e c t with a DNA chain encoding human G-CSF as described in US Pat. No. 4,810,643 de Souza. RhG-CSF is prepared as a 2.75-4 mg / ml solution in 100 mM sodium phosphate buffer, pH 6.0. The DTPA dianhydride and the t r i b u t i 1 f i n a (TBP, technical grade) is obtained from Aldrich (Milwaukee, Wl).

Preparation of the PIPA Conjugate: rhG-CSF Different amounts of the DTPA dianhydride are placed in dry, acid-washed analysis tubes (Meares, et al., J. P r o t C. C h e m., 3_, 215-228, (1984)). Then add two milliliters of the anhydrous chloroform (Aldrich Chemicals, Milwaukee, Wl), and the tube forms a vortex under a light stream of nitrogen gas to evaporate the chloroform and form a thin film of the DTPA dianhydride at the bottom of the tube. . RhG-CSF at a concentration of 2.75-4.0 mg / ml in the 100 mM sodium phosphate buffer, pH 6.0, pH 7.0, or pH 8.0 is added to the tubes coated with DTPA dianhydride at a final molar ratio 5: 1, 50: 1, or 500: 1 (DTPA: rhG-CSF) while stirring slightly. The aliquots of each sample are maintained and the volume of the sample is passed through a G50 rotation column as described; Penefske, H.S., Me t h o d s E n z y m o l. , 56, 527-530, (1979), in order to extract unconjugated DTPA.

Analysis of the DTPA Conjugate: rhG-CSF SDS-PAGE analysis.

The SDS-PAGE is performed on the samples treated in the G50 rotation column using ISS Miniplus gels at 17-27% (Nattick, MA). The samples are diluted with the non-reducing buffer, and 5 μg of the protein is reasonably loaded in each. The gels are run in a batch buffer system and stained with Coomassie Blue R-250 (Laemmli, UK, Na ture, 227, 680-685, (1970) .The SDS-PAGE analysis of the samples prepared with Priority is shown in Figure 1.

The reactions conducted with an initial pH of 7.0 (paths or tracks 5-7) or 8.0 (lanes or lanes 8-10) yield significant amounts of the higher molecular weight species compared to the apparent molecular weight observed for the untreated rhG-CSF (lane or lane 11 and 12). However, reactions with an initial pH of 6.0 (lanes or lanes 2-4) yield a simple detectable higher molecular weight species (corresponding to one of the species detected in samples of pH 7.0 and 8.0). The apparent molecular weight of these species suggests that this may be a protein dimer crosslinked with DTPA. With pH samples of 6.0, these higher molecular weight bands become less intense with the increase in the molar ratio of DTPA: rhG-CSF, and is virtually absent for the 500: 1 sample (path or track 4) .

HPLC with Size Exclusion The reaction mixture with an initial pH of 6.0, and the ratio of DTPA: rhG-CSF of 50: 1 is further analyzed by HPLC with size exclusion. A sample is analyzed in a pre-G50 rotation column and a post-G50 rotation column. HPLC is developed in a Waters Liquid Chromatography (Milliford, MA) equipped with a WISP 717 plus refrigerated unit at 5 ° C, and a 490E wavelength UV / Vis detector on the line with a Raytest Ramona LS radioisotope detector (Pittsburgh, PA). The empty volume between the UV / Vis detector and the radioisotope detector is 50 μl. For size-exclusion HPLC, the samples are analyzed with an isocratic mobile phase of the 0.1 M sodium phosphate buffer, 0.5 M NaCl, pH 6.9, on a Phenomenex BioSep S2000 column (Torrance, CA) eluted at 1.0 ml / min. at 25 ° C. Elution is monitored for absorbance at 280 nm and recorded by the Waters Millennium suite of programs on a PC computer.

As shown in Figure 2, the sample from the pre-G50 rotation column reveals two major peaks with the elution times of 8.65 and 9.53 minutes (Figure 2, line 2). The second major peak coelutes with the free DTPA and is almost eliminated in the sample from the post-G50 rotation column (Figure 2, line 3), which indicates the satisfactory extraction of unbound DTPA from the reaction mixture. The elution time of the remaining major peak is unchanged by means of the G50 rotation column and elutes slightly before the rhG-CSF without reaction (Figure 2, line 1). This peak represents rhG-CSF conjugated with monomeric DTPA. Thus, the behavior of the rhG-CSF conjugated with the DTPA in this size exclusion column allows to be redissolved from the unmodified rhG-CSF.

The above analysis shows that the initial pH and the molar ratio of DTPA: rhG-CSF can affect the formation of undesirable side reactions. By initializing the reaction in the buffer with an initial pH of 6.0, the formation of the products resulting from such side reactions (e.g., cross-linked proteins) is gradually reduced.

EXAMPLE 2 In this example the ability of DTPA: rhG-CSF conjugated to the 111In chelate is determined using HPLC by analytical cation exchange and thin layer chromatography. The conjugate is then analyzed to determine the mass of the conjugate (with and without chelated indium), the stoichiometric molar ratio of DTPA to rhG-CSF, and the location of the conjugated DTPA portion in the rhG-CSF.

Preparation of DTPA Conjugate: rhG-CSF In this example, the analysis is conducted on a DTPA conjugate: rhG-CSF prepared using an initial pH of 6.0, and a molar ratio of 50: 1 of DTPA dianhydride: rhG-CSF as described in Example 1. Without However, instead of passing the sample through a G50 rotation column, preparative cation exchange chromatography is performed using a high-resolution Pharmacia Hi-Load SP-Sepharose column, 16/10, with strong cation exchange. (Pharmacia, Sweden). The separation is carried out at 5 ° C by means of a Pharmacia FPLC system equipped with a 50 ml injection loop. The column is equilibrated in Shock Absorber A (20 mM sodium acetate, pH 5.4) and elution is carried out with a 0-40% Shock Absorber B gradient (20 mM sodium acetate, 0.5 M NaCl, pH 5.4 ) about 180 minutes at 1.0 ml / minute. The elution is monitored for absorbance at 280 nm and recorded.

The reaction mixture (initial pH of 6.0, molar ratio of DTPA dianhydride: rhG-CSF of 50: 1) originally containing 20 mg of rhG-CSF is diluted to 50 ml with Milli-Q water and applied directly to the Hi-Load SP-Sepharose column. A peak representing approximately 13% of the integrated peak areas (Figure 3, peak 2) coelute with the unreacted control of the rhG-CSF (Figure 3, faded line). This indicates that approximately 13% of the rhG-CSF remains unchanged. A peak eluted between 120 and 130 minutes accounted for 84% of the total eluted protein (Figure 3, peak 1). The change in this material to elute in a concentration is in accordance with an increase in the negative charge in the protein by means of conjugation with DTPA.

This step of simple and efficient chromatography produces a homogeneous and well-defined product with the unbound DTPA and the conjugated rhG-CSF separated from the DTPA conjugate: rhG-CSF purified.

Analysis of the DTPA Conjugate: rhG-CSF HPLC by Analytical Cation Exchange HPLC by analytical cation exchange is developed with the mobile phases of Shock Absorber A (20 mM sodium acetate, pH 5.4), and Shock Absorber B (20 mM sodium acetate, 0.5 M NaCl, pH 5.4) on a Tosohaas SP column -5PW, 7.5 X 7.5 mm column (Montgomery, PA) using the Waters HPLC system. The column is equilibrated with mobile phase A, and the separation is carried out at 25 ° C with a linear gradient of 1% B / min over 30 minutes at 1.0 ml / minutes. The separation is detected by monitoring the absorbance at 220 nm, and where applicable, with the radioisotope detector. For samples containing indium, 1 mM EDTA is added to Buffer A before adjusting the pH to 5.4.

The rhG-CSF and the DTPA conjugate: rhG-CSF are preincubated with 111In for 15 minutes. An excess of crude indium is then added (IN: protein, 2: 1, mol / mol), and the samples analyzed by means of HPLC with analytical cation exchange are described above. For the DTPA conjugate: rhG-CSF (Figure 4 lines 1 and 4, solid), 99.5% of the activity of 111In coeluted from the column by cation exchange with the protein, since the radioactivity co-detected was not detected. eluted with the unmodified rhG-CSF (Figure 4, lines 2 and 3, faded) indicating the chelation of 111In by the conjugate of DT PA: r G-CSF, and the absence of ligation of the 111 In by the rhG-CSF without modifying The effect of metal chelation on the HPLC analysis by analytical cation change of the DTPA conjugate: rhG-CSF is depicted in Figure 5. Elution of the rhG-CSF (line 1), the DTPA conjugate: rhG -CSF (line 2), and the conjugate pre-incubated with excess of I n C 13 (In: conjugate, 10: 1, mol / mol, line 3), is monitored for absorbance at 220 nm. The DTPA conjugate: rhG-CSF elutes at a lower salt concentration than the unmodified rhG-CSF. The chelated conjugate then elutes at a slightly higher salt concentration than the non-chelated conjugate but still at a lower salt concentration than the unmodified rhG-CSF. The characteristic retention time of the DTPA: rhG-CSF with and without the chelated metal can be used to monitor the metal contamination of the conjugate preparation.

In addition, this analysis can be used to monitor the classification or marking of the metal of the conjugate.

Thin layer chromatography (TLC The TLC is developed as described previously (Meares et al., J. P r o t C h e m., 3_, 215-228, (1984)) with slight modifications. A solution of the concentrated indium base containing InCl3 with a trace of 111 In is prepared in 10 mM HCl, and is used to prepare the following samples: (1) indium added to 100 mM sodium phosphate, pH 6.0; (2) indium 10 nmol added to DTPA 20 nmol in 20 mM sodium acetate, pH 5.4; (3) 10 nmol of indium incubated with rhG-CSF 2 nmol at room temperature for 10 minutes, followed by the addition of 20 nmol of DTPA in 20 mM sodium acetate, pH 5.4, and (4) 10 nmol of indium incubated with 2 nmol of rhG-CSF conjugated with DTPA at room temperature for 10 minutes, followed by the addition of 20 nM DTPA in 20 mM sodium acetate, pH 5.4. One μl of each sample (containing 0.1 nmol of indium) is placed on the silica gel of 250 μm thickness (60 A) on the glass support (Whatman, Clifton, NJ). The TLC plate is developed using 10% ammonium acetate (w / v) in H20 of s t i 1 ada: me t ano 1 (1: 1, v / v) as the solvent. The developed plate is then analyzed using a Phosphorus Imaging Processor by Molecular Dynamics (Molecular Dynamics P o p h o r I ma g e r) (Sunnyvale, CA).

The stoichiometric molar ratio of DTPA to rhG-CSF is determined as described hereinabove. The chelation of 111In by means of DTPA results in the migration of all to radioactivity from near the front solvent (Figure 6, compare lanes or lanes 1 and 2). Incubation of 111 In (10 nmol) with the DTPA conjugate: rhG-CSF (2 nmol), followed by the addition of DTPA, results in the retention of a portion of the radioactivity at the origin (Figure 6, lane or lane 4). ). The in-line graphs of the individual tracks or tracks are generated and the integration of the areas of the peak of the path or line 4 reveals 18% of the radioactivity remaining at the origin. The remaining 1t1In bond is cleaned by the addition of DTPA and migrates near the front of the solvent. Thus, approximately 1.8 nmol of 111In is bound by 2 nmol of the DT P A conjugate: r h G - C S F, which indicates a molar ratio of DTPA: hG-CSF of 0.9. The unmodified rhG-CSF does not retain the radioactivity at the origin, indicating the absence of ligation of the 111In (Figure 6, lane or lane 3).

Mass Spectrometry The mass spectrometry of d e s p r i o n / i o n i z a c i o n with the laser supported by the matrix (MALDI-MS) is developed with a mass spectrometer Kompact MALDI III (Kratos Analytical, Ramsey, NJ) adapted with a standard nitrogen laser 337 nm. The spectrum is recorded with the analyzer in linear mode with an acceleration voltage of 20 kV. An aliquot of the sample containing 15 pmol of the protein and 1.0 μl of the alpha-cyano-4-1 d or x i c i n a m i c o are mixed in the sample reasonably from the research slip and allowed to air dry. The laser fluence of the instrument is set to 30 (adjustable over a relative scale of 0-100).

The mass of the DTPA conjugate: rhG-CSF is determined by MALDI-MS (Figure 7). The spectrum obtained reveals the multiple ions also charged to the species mo n o p r o t o n a d s s. The mass obtained by the average of the peak series is 19,171.7 (± 7.3) Da. The MW calculated for a simple conjugate of the DTPA to the rhG-CSF is 19,170.8 Da. Therefore, in general agreement with the analysis of the TLC, the mass observed indicates a molar ratio of DTPA to rhG-CSF of 1: 1 for the DTPA conjugate: rhG-CSF.

Mass spectrometry by ion atomization The ion atomized mass spectrometry is performed with a Perkin-Elmer Sciex API III mass spectrometer (Norwalk, CT) equipped with an ion atomizing interface by means of the flow injection method. The samples are diluted in a nominal form (50: 50: 0.1, V / V) and the flow injected in the same solvent flowing at 25 μl / minute. The holes are fixed at 70 V, and the mass spectrometer operates in the Ql mode.

The mass of the DTPA conjugate: rhG-CSF with the chelated indium is determined as described above. The analysis of the conjugate, preincubated with the saturation indium (In: conjugate, 10: 1, mol / mol), produces a series of peaks with values that differ m / z. These multiple series of charged ions, amounting to multiple protonation of the protein, are unwound to produce the MW spectrum shown in Figure 8. The measured mass of the conjugate with the chelated indium is 19.286 (± 1.7) Da, which is in agreement with the calculated mass of 19.285.6 Da. For the rhG-CSF, the measured mass is 18,798 (± 1.8) Da (Figure 9), according to the calculated mass' of 18,798.5 Da.

Peptide Mapping For the analysis of the peptide, approximately 0.5 mg of the rhG-CSF or DTPA: rhG-CSF are dried in a vacuum exposure time, reconstituted in 100 μl of 8M Urea and sonicated for 10 minutes. After sonication, 10 μl of 1 M Tris-HCl, pH 8.5 and 2.5 μg of EndoLys-C (Wako Chemicals, Richmond VA) of 1 mg / ml of the concentrated base solution in Tris HCl 10 mM, pH 8.5 are added. The total volume is adjusted to 200 μl with distilled water, and the proteolytic digestion is carried out for 7 hours at room temperature. Following hydrolysis with EndoLys-C, disulfide bonds are reduced simultaneously with 5 μl of 80 mM TBP and alkylated with 10 μl of 40 mM ABD-F (final concentration 2 mM) as described; Kirley, T.K. , A n a l. B i or c h e m. , 180, 231-236, (1989). Immediately after reduction and alkylation, the generated peptides (200 μl) are injected directly into an inverted phase HPLC column C of 300 A pore size (Separation Group, Vydac, Hesperia, CA) equilibrated with the Solvent A (0.1% TFA in distilled water). Peptide analysis is developed using a Waters HPLC system consisting of two pumps 510, a WISP 712 autoinjector, and a 481LC spectrophotometer, all controlled through a system interface module by means of the system software package, Maximum The generated peptides are eluted with a linear gradient of Solvent B at 3-76% (0.1% TFA, 95% acetonitrile) over 115 minutes. Elution is monitored for absorbance at 215 nm. The individual peptides of the standard peptide map rhG-CSF are collected and identified by analysis of amino acid composition and the N-terminating chain as described; Souza et al., S c i e n c, 232, 61-65, (1986).

Fragments of the unmodified G-CS "F" peptide and DTPA: rhG-CSF are prepared and analyzed as described above.A peak eluting from the unmodified rhG-CSF sample in 60 minutes is found absent from the conjugate sample of the DTPA: rhG-CSF (Figure 10) Elution of the material in this peak is determined by means of the analysis of the amino acid composition and the N-terminating chain to be residues 17 of the N-terminus of rhG-CSF Thus, the fragment corresponding to the peptide with the N terminus of the DTPA conjugate: rhG-CSF is modified, producing a double division peak in a new partial form eluting at 62 minutes. of the peptide from each of these partially separated peaks by means of mass spectrometry reveals that the first peak has the expected mass of the peptide with the N terminus with the conjugated DTPA, while the mass of the second peak of the material suggests that he Conjugated peptide is contaminated with iron. Therefore the mapping of the peptide indicates that the conjugated DTPA group is located in the 17 amino acids with N-terminus. These N-terminating peptides containing the N-terminus in threonine, three serine residues and one residue of lysine The separation of the peptide by means of Endolys-C indicates that the lysine is unchanged. The acylation of threonine or serine residues is highly different at the pH of 6.0. The DTPA conjugate: undigested rhG-CSF is subjected to the chain by the end of N reveals > 99% of the blocked N terminus, which indicates the simple portion of DTPA in the protein is conjugated to the N-terminus.

Isoelectric focus The isoelectric focus is developed using the Novex pH gels of 3-10 (San Diego, CA) with a pl performance range of 3.5 - 8.5. The samples are diluted 1: 1 with the buffer of the sample, and 5 μg of the protein is loaded on each path. The gels are run at constant voltages of 100 V for 1 hour, 200 V for 2 hours, and then 500 V for 0.5 hour. All fixing, staining and destintion procedures are given for the manufacturer's specifications.

The DTPA conjugate: rhG-CSF reveals a simple upper band of pl 4.9 that follows the isoelectric focus (Figure 11, path or track 3). Preincubation of the conjugate with the excess of InCl3 (I n: c on j ugado, 10: 1, mol / mol) displaces the band to the pl of 5.3 (Figure 11, lane or lane 2). The pl values of the conjugate, both with or without indium, are lower than that of the rhG-CSF, pl 6.0 (Figure 11, lane or lane 1). The conjugation of DTPA, the concomitant with the loss of the free amino group with the N-terminus, substantially decreases the pl of the rhG-CSF. In addition, the chelated indium increases the pl of the conjugate slightly. The characteristic isoelectric points of the DTPA conjugate: rhG-CSF with and without the chelated metal can also be used to monitor the contamination of the metal and the classification or marking of the metal of the conjugate preparation.

The above data show that: (1) the DTPA conjugate: rhG-CSF is capable of chelating 111In; (2) the stoichiometric molar ratio of DTPA to rhG-CSF is approximately 1.0 for the DTPA conjugate: rhG-CSF; (3) the conjugation of DTPA is specific to the N termination of the rhG-CSF; and (4) the conjugation of DTPA to rhG-CSF decreases the pl of rhG-CSF.

EXAMPLE 3 In this example, the circular dichroism analysis is used to study the effects of the secondary structure of the rhG-CSF that results from the conjugation of a chelating group to the N-terminus of the rhG-CSF.

The Spectrum of Circular Dichroism (CD) is obtained with an e s p e c t o r t o r t h e t t J Jcoco J-720 (Japan S p e c t r o s c o c i c Co., LTD., Tokyo, Japan). The samples (0.078 mg / ml of the protein) are analyzed at 10 ° C in 20 mM sodium acetate, pH 5.4. The CD spectrum of the DTPA conjugate: rhG-CSF covers the unmodified rhG-CSF (Figure 12), each revelation of the minimum ellipticity at 208 nm and 222 nm. The addition of the excess indium to saturate all the chelating sites in the conjugate (In: conjugate, 10: 1, mol / mol) does not change the overall shape of the spectrum, which still causes a slight reduction (about 5%) in the alpha helicity. Therefore, the secondary structure (at pH 5.4) is shown here not to be influenced by the conjugation of a chelating group to the N-terminus.

EXAMPLE 4 In this example, the effects of DTPA conjugation and subsequent chelation of the indium on the biological activity of rhG-CSF are determined.

The peripheral counts of WBC in the hamsters by rhG-CSF, the conjugate of the DTPA: rhG-CSF, and the conjugate with the chelated indium are evaluated after the subcutaneous injection of 100 μg / kg of the rhG-CSF in the hamsters (Figure 13). The animals are sacrificed at the indicated time intervals, and the collected blood samples are analyzed using a counter for Sysmex F800 microcells.

The injection of the conjugate (Figure 13, (?)) Induces the level of peripheral WBC counts in a manner similar to the unmodified rhG-CSF (Figure 13, (o)). Injection of the preincubated conjugate with excess indium (In: conjugate, 10: 1, mol / mol) (Figure 13, (0)) also induces a similar response, with the maximum WBC levels reached in 24 to 36 hours at post - injection. Thus, the conjugation of DTPA and rhG-CSF does not significantly alter the activity observed in the rhG-CSF, and in addition the activity of the conjugate is unchanged after the chelation of the indium.

EXAMPLE 5 In this example, the conjugation reaction described above is carried out on the related growth factor, interleukin (IL-2). The ability of the DTPA conjugate: IL-2 to chelate 111In is evaluated using HPLC by exchange of the cation as described above. In addition, the stoichiometric molar ratio of DTPA: IL-2 is determined as well as the distribution of the DTPA portion in IL-2.

IL-2 is produced using recombinant DNA technology where E cells. c or l i are transfected with a DNA sequence encoding IL-2 as described in European Patent No. 0136489 (Souza et al.). IL-2 is prepared as a 1.82 mg / ml solution in the 100 mM sodium phosphate buffer, pH 6.0. The DTPA dianhydride and the t r i u t i 1 f i n a (TBP, technical grade) are obtained from Aldrich (Milwaukee, Wl). The conjugates are prepared as described in Example 2 above.

Analysis of the DTPA Conjugate: IL-2 HPLC by Analytical Cation Exchange HPLC by exchange of the analytical cation is developed as described above. For the DTPA conjugate: IL-2 (Figure 14, lines 1 and 4, solid), 99.5% of the radioactivity of 111In cools off the column by exchange of the cation with the protein, since it does not coelute the radioactivity detectable with the Unmodified IL-2 (Figure 14, lines 2 and 3, vanished), which indicate the chelation of 111In by the DTPA conjugate: IL-2, and the absence of ligation of the 111 In for the unmodified IL-2 The effects of chelation of the metal in the HPLC analysis by exchange of the analytical cation of the DTPA: IL-2 conjugate is illustrated in Figure 15. The elution of IL-2 (line 1), the DTPA conjugate: IL- 2 (line 2), and the conjugate pre-incubated with excess lnC3 (I n: conjugate, 10: 1, mol / mol, line 3) is monitored for absorbance at 220 nm. The DTPA conjugate: IL-2 eluting at a lower salt concentration than unmodified IL-2. The chelated conjugate eluting at a salt concentration slightly higher than the uncharged conjugate, but still at a lower salt concentration than that of unmodified IL-2. As is the case with the analysis of the rhG-CSF above, the retention times characteristic of the DTPA conjugate: IL-2 provides a useful method of monitoring the contamination of the metal and the classification or marking of the metal of the conjugate.

Thin Layer Chromatography (TLC TLC is developed as described above in order to determine the ability of the DTPA conjugate: IL-2 to chelate 111 In, and to determine the stoichiometric molar ratio of DTPA to IL-2.

As shown in Figure 16, chelation of 111In by DTPA results in the migration of all radioactivity near the solvent front (Figure 16). (10 nmol) with the DTPA conjugate: IL-2 (2 nmol), followed by the addition of DTPA, results in the retention of a portion of the radioactivity of the origin (Figure 16, lane or lane 4). The line graphs of the individual tracks or tracks are generated and the integration of the areas of the peak of the path or track 4 reveal 18% of the radioactivity remaining at the origin. The unlinked 111In remnant is cleaned with the added DTPA and migrates near the solvent front. Thus, approximately 1.8 nmol of 111 In is bound by means of 2 nmol of the DTPA conjugate: IL-2 which indicates a molar ratio of DTPA to I L-2 of 0.9: 1. The unmodified IL-2 does not retain the radioactivity at the origin, which indicates the absence of the 111In ligature (Figure 16, lane or lane 3). 3. Peptide mapping.

Peptide analysis is performed on the DTPA: IL-2 conjugate in order to determine the location of the conjugated DTPA portion in IL-2. Peptide fragments are prepared as described above.

As shown in Figure 17 (arrow), a peak eluting from the unmodified IL-2 sample in approximately 36 minutes is absent from the sample of the DTPA: IL-2 conjugate. The material eluting at this peak is determined by the analysis of the amino acid composition and the N-terminating chain to be the N-terminating peptide of IL-2. Therefore, the fragment of the peptide with the corresponding N-terminus of the DTPA: IL-2 conjugate is modified, resulting in a partial separation of the peak in 40 minutes. As is the case with the rhG-CSF, the peptide mapping indicates that the conjugated DTPA group is located at the N-terminus.

These data demonstrate that the DTPA: IL-2 conjugate is capable of chelating 111In, that the stoichiometric molar ratio of DTPA to IL-2 is approximately 1.0, and that DTPA is conjugated specifically located at the N-terminus of the IL-2.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (20)

1. A composition characterized in that it comprises a chelating agent, a protein, and a metal cation, the chelating agent binds the metal cation and the conjugate located specifically at the N-terminus of the protein.

2. The composition according to claim 1, characterized in that the metal cation is radioactive.

3. A composition according to claim 1 or 2, characterized in that the chelating agent is the dicyclic anhydride of diethylenetriaminepentaacetic acid.

4. A composition according to claim 2, characterized in that the radioactive metal cation is selected from the group consisting of gallium 67, indium 111 and technetium 99m.

5. A composition according to claim 1 or 2, characterized in that the protein is selected from the group consisting of G-CSF, GM-CSF, M-CSF, interferons (alpha, beta, and gamma), the i nt er 1 euci as (1-14), eriropoietin (EPO), fibroblast growth factor, stem cell factor, nerve growth factor, NT3, megalcarcum growth and development factor (MGDF), growth factor of derived platelets (PDGF) and tumor growth factor (alpha, beta).

6. A composition according to claim 5, characterized in that the protein is G-CSF.

7. A composition according to claim 5, characterized in that the protein is I L-2.

8. A composition characterized in that it comprises the dicyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA); the rhG-CSF; and indium 111, DTPA binding to indium 111 and conjugate located specifically at the N terminus of rhG-CSF.

9. A composition characterized in that it comprises the dicyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA); rhIL-2; and indium 111, DTPA which binds indium 111 and conjugate specifically located at the N terminus of rhIL-2.

10. A method for the preparation of a classified or labeled protein, the method characterized in that it comprises: (a) the reaction of a chelating agent with the protein at a sufficiently acid pH to selectively activate the a-amino group at the amino terminus of the protein; (b) the separation of the conjugated protein from the unconjugated protein; (c) the addition of a metal cation to the conjugate; Y (d) obtaining the protein classified or labeled.

11. A method according to claim 10, characterized in that the pH is 6.0.

12. A method for the preparation of G-CSF, classified or labeled, the method characterized in that it comprises: (a) the reaction of a chelating agent with G-CSF at a sufficiently acidic pH to selectively activate the a-amino group at the amino terminus of G-CSF; (b) the separation of the conjugated G-CSF from the unconjugated G-CSF; (c) the addition of a metal cation to the conjugate; and (d) obtaining the classified or marked G-CSF.

13. A method according to claim 12, characterized in that the pH is 6.0.

14. A method according to claim 13, characterized in that the chelating agent is DTPA.

15. A method for the preparation of labeled or labeled IL-2, the method characterized in that it comprises: (a) the reaction of a chelating agent with IL-2 at a sufficiently acidic pH to selectively activate the a-amino group at the amino terminus of IL-2; (b) the separation of conjugated IL-2 from unconjugated IL-2; (c) the addition of a metal cation to the conjugate; and (d) obtaining the labeled or labeled IL-2.

16. A method according to claim 15, characterized in that the pH is 6.0.

17. A method according to claim 16, characterized in that the chelating agent is DTPA.

18. A pharmaceutical composition: characterized in that it comprises: (a) a substantially homogeneous preparation of the recombinant human G-CSF, the recombinant human G-CSF consisting of a portion of the chelating agent with the bond of the chelated metal cation, conjugated to a portion of the recombinant human G-CSF only at the N-terminus thereof by means of an amide bond; and (b) a pharmaceutically acceptable diluent, adjuvant or carrier.

19. A pharmaceutical composition characterized in that it comprises: (a) a homogeneous preparation in substantial form of recombinant human IL-2, recombinant human IL-2 consisting of a portion of the chelating agent with the bound chelated metal cation, conjugated to a portion of recombinant human IL-2 only at the N-terminus thereof by means of the amide bond, and (b) a pharmaceutically acceptable diluent, adjuvant or carrier.

20. A composition according to claim 1 or 2, for use as an agent for the detailed production of diagnostic images.