EP0140968A1 - Zusammensetzung zur induktion von suppressor-t-zellen und verfahren - Google Patents

Zusammensetzung zur induktion von suppressor-t-zellen und verfahren

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Publication number
EP0140968A1
EP0140968A1 EP84902121A EP84902121A EP0140968A1 EP 0140968 A1 EP0140968 A1 EP 0140968A1 EP 84902121 A EP84902121 A EP 84902121A EP 84902121 A EP84902121 A EP 84902121A EP 0140968 A1 EP0140968 A1 EP 0140968A1
Authority
EP
European Patent Office
Prior art keywords
peptide
cells
composition
amino acid
differentiation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP84902121A
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English (en)
French (fr)
Inventor
Michael E. Osband
Ellen Beth Cohen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boston University
Original Assignee
Boston University
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Filing date
Publication date
Application filed by Boston University filed Critical Boston University
Publication of EP0140968A1 publication Critical patent/EP0140968A1/de
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • This invention relates to therapeutic and diagnostic peptides.
  • Thymosin disclosed by Goldstein et al., U.S. Patent 4,297,276, is derived by a process which includes an extraction step with acetone.
  • the Thymosin products disclosed in U.S. Patent 4,297,276 are characterized by amino acid residues of 50 or 43.
  • Another form of Thymosin has a molecular weight of about 12,200 and is characterized by 108 amino acid residues.
  • the product, thymopoetin, disclosed by Goldstein in U.S. Patent 4,120,951 has a molecular weight of approximately 6,000 and contains 53 amino acids.
  • Thymic Humoral Factor disclosed by Trainin has a molecular weight of approximately 3,100 and is composed of 32 amino acids.
  • the invention features a composition characterized in that it is capable of inducing the differentiation of suppressor T-cells in vivo, it is substantially free of impurities which cause undesirable side effects when administered to an animal, and it is stable both in solution and dry.
  • the composition is made by the steps of homogenizing thymus gland (preferably calf thymus) to produce a homogenate, fractionating the homogenate to yield a first fraction containing compounds having molecular weights less than about 10,000 daltons, fractionating the first fraction into a plurality of fractions using ammonium sulfate fractionating one of the fractions to yield a plurality of fractions having different electrical charges, and recovering .the fraction exhibiting the capability of inducing the differentiation of suppressor T-cells in vivo and i n vitro.
  • the above composition contains a peptide (present as approximately 20% of the composition, w/w), which is characterized in that it is capable of inducing the differentiation of hui ⁇ an suppressor T-cells, it contains seventeen to twenty amino acids, it has the amino acid composition Trp, Asn, Asp 1 -2 , Ser 3 , Glu 2-3 , Gly 1-2 , Ala 2 , Val, lle. Leu, Tyr, Phe, Lys, and it has a molecular weight of between about 1,700 and 2,400 daltons.
  • This peptide is designated peptide A2.
  • the invention also features A2 which is substantially pure, as shown by SDS PAGE and high performance liquid chromatography (HPLC).
  • the invention also features a smaller peptide (which may oe a breakdown product of A2, as will be explained in more detail below), designated peptide B1, which is also provided in substantially pure form, as shown by SDS PAGE and HPLC, and which is characterized in that it is capable of inducing the differentiation of human suppressor T-cells in vivo and has the amino acid composition Trp, Asn, Ser 3 , Gly, Ala, Lys, the C-terminal amino acid being Lys.
  • peptide B1 which is also provided in substantially pure form, as shown by SDS PAGE and HPLC, and which is characterized in that it is capable of inducing the differentiation of human suppressor T-cells in vivo and has the amino acid composition Trp, Asn, Ser 3 , Gly, Ala, Lys, the C-terminal amino acid being Lys.
  • Peptides A2 and B1 can be admixed with pharmaceutically acceptable carrier substances to form therapeutic compositions.
  • the peptides and compositions of the invention can be used to treat diseases, e.g., histiocytosis-x, in which T-cell dysfunction is a causative factor, and particularly diseases in which there is a deficiency in the number of histamine H2 receptor-bearing suppressor T-cells (H2R+ cells).
  • diseases e.g., histiocytosis-x, in which T-cell dysfunction is a causative factor, and particularly diseases in which there is a deficiency in the number of histamine H2 receptor-bearing suppressor T-cells (H2R+ cells).
  • the Figure is a flow diagram of a procedure used to purify A2 and B1.
  • A2 and B1 can be purified to homogeneity from calf thymus, and the pure peptides used in therapeutic compositions, or a composition containing at least 20% A2, w/w, can be prepared from calf thymus and used therapeutically without further purification.
  • the first step is to homogenize fresh calf thymus in physiological buffer solution, at a pH between about 6 and about 8. The homogenate then is centrifuged and a supernatant is recovered. The supernatant is then concentrated by filtration and lyophilization prior to precipitation with ammonium sulfate. Alternatively, lyophilization is omitted, and the filtrate is saturated with ammonium sulfate (100%). Since protein precipitation is dependent upon protein concentration, the concentration of ammonium sulfate used will vary depending upon whether the lyophilization step is included.
  • the above procedure is as follows. First, the supernatant from the centrifugation of the calf homogenate is filtered in order to recover a filtrate having a molecular weight less than about 10,000 daltons.
  • the Pellicon ultrafiltration membrane available from Millipore Corporation of Bedford, Massachusetts is suitable for this purpose.
  • the filtrate having a molecular weight less than about 10,000 daltons then is subjected to ammonium sulfate fractionation.
  • the filtrate is admixed with ammonium sulfate to make a 40% solution to precipitate a selected portion of the filtrate.
  • the precipitate is separated out, such as by centrifugation, and the solution is recovered.
  • the recovered solution then is brought to a 70% aqueous solution of ammonium sulfate to precipitate another portion of the filtrate, which contains A2.
  • the latter is recovered and dissolved in distilled water.
  • the active material is present in the supernatant in a dilute state and is saturated with ammonium sulfate (100%).
  • the A2-containing material in solution can next be further purified according to one scheme to yield a composition which contains about 20%, w/w A2, or can be subjected to an alternate purification scheme resulting in substantially pure A2 and substantially pure B1.
  • the material from the previous step is mixed with a buffer to form a solution having a pH between about 7.0 and 7.4, and the solution is then desalted by passing it through a filtration column such as a gel column, e.g., a Sephadex G-10 column.
  • a filtration column such as a gel column, e.g., a Sephadex G-10 column.
  • the extract then is passed through a chromatographic column of a DEAE Sephadex ion exchange resin which does not degrade the protein material and which is equilibrated with a salt solution used to dissolve the thymic extract.
  • the extract is separated into components within the column.
  • the column then is eluted in a conventional manner with an aqueous sodium chloride solution to recover the individual fractions.
  • the extract fractions rich in the proteinaceous material that induces H2R+ suppressor T-cells then is recovered by identifying the fraction having this desired activity; this is the fractionrecovered by eluting with 0.1-0.2 molar sodium chloride gradient.
  • the active fraction contains A2 as well as non-A2 proteinaceous materials which do not adversely affect the stability of the A2. That is, the extract separated within the column is not subjected to enzymatic or proteolytic degradation, in contrast to the active material in its natural state or in the thymus gland.
  • the active fraction is further fractionated in an HPLC chromatographic separation column such as the C 18 column available from Waters Associates, Inc., Milford, Massachusetts.
  • the bound protein then is eluted with a dual solvent gradient comprising 20 millimolar aqueous sodium phosphate (Solvent A) and 80% acetonitrile/20% water, Vol/Vol (Solvent B).
  • the active fraction containing about 20% A2, is recovered by passing a continuous solvent gradient through the column and recovering the protein fraction which is eluted with 20-50 vol % of Solvent B. Purification of A2 and B1 to Homogeneity
  • the flow diagram of the Figure illustrates the scheme used to purify A2 and B1 (as well as two other peptides, A1 and B2) to homogeneity.
  • the A2-containing solution from the saturated ammonium sulfate solution, above, is mixed with a buffer to form a solution having a pH between about 7.0 and 7.4, which is passed through a pnenyl Sepharose column to separate initially those constituents of the solution that bind from those that do not.
  • the less polar fraction of the bound material, containing A2 is further purified by fractionating on DEAE Sephadex and recovering the bound material and subjecting that to HPLC, recovering the active fraction, and then subjecting that fraction to an additional HPLC step to yield homogeneous A2.
  • composition containing about 20% A2, w/w is prepared as follows.
  • the filrate is lyophilized to dryness and reconstituted with Phosphate Buffered Saline to a concentration of 50 mg protein/ml, and is then subjected to differential precipitation with 40-70% ammonium sulfate.
  • the filtrate 150 ml is first mixed with ammonium sulfate to a final concentration of 40% and stirred for 120 minutes at room temperature to obtain a precipitate, which is removed by centrifugation; 125 ml of the supernatant are recovered.
  • the supernatant is mixed with additional ammonium sulfate to a final concentration of 70% to precipitate another fraction. This fraction is recovered by centrifugation and dissolved in 10 ml distilled water.
  • the dissolved material above is desalted on a G-10 column (2.5 cm x 10.5 cm, 55 ml) in 10 mM sodium phosphate, pH 7.2, and 1.0 ml fractions are collected. Total pooled volume is 5.0 ml. The 280 nra absorbance of the pooled material is 0.970.
  • the active fraction from the DEAE column [SUPR-I-15 (III) ] is applied to a Waters C 18 HPLC chromatographic column, which is then eluted with a gradient of 80% CH 3 CN (Solvent B) into 20 mM potassium phosphate (pH 6) (Solvent A).
  • the active fraction is that obtained with 20-50 vol % Solvent B. This fraction contains about 20% A2, w/w.
  • Homogeneous A2 and B1 are prepared, by the purification scheme illustrated in the Figure, as follows. 5000 grams of fresh excised calf thymus are admixed with 2500 ml of 50 mM ammonium bicarbonate buffer at pH 7 and homogenized in a food processor. The homogenate is then centrifuged and 3000 ml of supernatant are recovered by centrifugation. The supernatant is then filtered through a coarse fiber bag to remove fat and other particulate matter and the filtrate concentrated by filtration in a Pellicon ultrafilter to obtain a filtrate having a molecular weight less than about 10,000 daltons. Ammonium sulfate was added to the filtrate to obtain a saturated solution (100%).
  • the solution is passed through a phenyl Sepharose column (5 cm x 20 cm, 390 ml) in 10 mM sodium phosphate to separate the less polar fraction, containing A2, from the more polar fraction, containing Bl.
  • the less polar fraction is collected (50 ml) and loaded on a DEAE Sephadex chromatographic column (2.5 x 20 cm, 100 ml) in 10 mM sodium phosphate, pH 7.2, and fractionated using a sodium chloride gradient.
  • the active fraction then is passed through a Waters C 18 HPLC chromatographic column, and eluted with a gradient of 80% acetonitrile (Solvent B) into 20 mM phosphate (pH 6.8) (Solvent A).
  • the active fraction obtained with 20% to 50% Solvent B is mixed with 6.7 mM (TFA), pH 2.2.
  • the resultant mixture is passed through a Waters Micro-Bondopak C 18 HPLC chromatographic column in 0.05% v/v TFA and a linear gradient is run to 100% 4:v/v acetonitrile-water (Solvent B).
  • the active fraction obtained with 45% to 70% solvent B is homogeneous A2.
  • B1 is recovered from the more polar fraction bound to the phenyl Sepharose chromatographic column. This fraction is passed through a CM-Sephadex column in order to bind the peptide Bl-rich fraction to the column (2.5 cm x 20 cm, 100 ml). The bound fraction then is eluted with a sodium chloride gradient and the 0.1 to 0.2 molar fraction is recovered. This fraction then is mixed with 20 mM phosphate, pH 6.8 and passed through a C 18 HPLC column. The column is eluted with an acetonitrile solvent gradient and the 30% to 50% fraction is recovered. This fraction is passed through an HPLC C 18 column as above with the active fraction eluted between 30% and 55% Solvent B.
  • Table 1 gives relative abundance in thymus, relative potency, and fraction of suppressor activity expressed, for peptides A2 and B1 (as well as for peptide B2, which is the same molecule as is described in Bach et al. U.S. Pat. No. 4,133, 804, and peptide Al, which is not included in the present invention).
  • A2 is 1.5 times as potent as B2, w/w, and 3.25 times as potent as B2, mol/mol.
  • Amino acid analysis of A2 indicates that it contains between seventeen and twenty, inclusive, amino acids, and has a molecular weight of between about 1,700 and 2,400 daltons.
  • the amino acid composition of A2 is: Trp, Asn, Ser 3 , Gly 1-2 , Ala 2 , Lys, Asp 1-2 , Glu 1-2 , Val, lle. Leu, Tyr, Phe.
  • Amino acid analysis of B1 indicates that it contains eight amino acids and has a molecular weight of 877 daltons.
  • the amino acid composition of B1 is: Trp, Asn, Ser 3 , Gly, Ala, Lys.
  • Both A2 and B1 appear to be N-terminally blocked by an acetyl group. (This is reflected in the molecular weight determination of B1.)
  • B1 is a breakdown product of A2.
  • the model is illustrated below.
  • Peptide B 1 Table 2 gives activity and purification data for the thymic fractions obtained at various stages of the purification scheme described above and illustrated in the Figure. Activity was measured by the in vivo induction of functionally active suppressor cells in a raurine model of delayed-type hypersensitivity. As shown in Table 2, B1 purified to homogeneity (O.C.H 2 ) has almost 10,000 times the activity, w/w, of the B1-containing fraction (O 1 .C) prior to HPLC. Similarly, A2 purified to homogeneity (O 2 .H 1 -II.H 2 ) has almost 100.000 times the activity of the A2-containing fraction (O 2 ) prior to HPLC.
  • a therapeutic composition for in vivo use is prepared by diluting, in a physiologically compatible buffer such as normal saline, either pure A2, pure B1, a mixture of A2 and B1, or an incompletely purified A2-containing composition containing between 20% and 99%. w/w, A2.
  • the patient to be treated is then skin-tested prior to the first treatment to detect any acute hypersensitivity.
  • the composition is given daily, e.g. as an intramuscular injection at six rotating sites (arm, legs, buttocks); treatment is continued until normal numbers of suppressor cells have been induced, until the condition being treated improves significantly, or until a significant allergic reaction occurs. Treatment is resumed in the case of a relapse.
  • the concentration of protein in the therapeutic composition depends on the percentage of A2 and/or B1 making up the protein.
  • the daily dosage will range between about 0.1 and 2.0 ng protein/kg body weight, with a dosage of about 0.33 ng protein/kg body weight being preferred.
  • Pure A2 and B1, or a mixture of the two, will be administered in daily dosages of about 0.001 and 0.5 ng protein/kg body weight, with a dosage of about 0.1 ng protein/ kg body weight being preferred.
  • the therapeutic compositions can be administered by any other medically acceptable method, e.g., intravenously, peritoneally, orally, nasally, or by timed-release implant.
  • the therapeutic compositions can be used to treat diseases involving suppressor T-cell dysfunction. and particularly diseases involving an insufficiency in the number of suppressor T-cells.
  • Induction of suppressor T-cells with the compositions of the invention can be useful in the treatment, for example, of graft-versus-host disease, in which lytic T-cells are an important factor.
  • the compositions can also be used therapeutically to inhibit mitogen response, to inhibit non-specific immunoglobulin synthesis, and to suppress delayed-type hypersensitivity reactivity.
  • Clinical conditions which, because they are associated with a deficiency of suppressor T-cells, include atopy/allergy, multiple sclerosis, rheumatoid arthritis, SLE, ulcerative colitis, and the immunosenescence associated with aging.
  • One class of patients who are candidates for treatment are those in which a manifestation of their disease is the presence of autocytotoxic lymphocytes. Such patients can be screened easily by incubating their autocytotoxic lymphocytes with the therapeutic composition for several hours; if the autocytotoxic reactivity is abrogated, there is a likelihood that the patient will benefit from treatment.
  • Histiocytosis-X is histiocytosis-X, actually a group of rare syndromes (Letterer-Siwe disease, Hand-Schuller-Christian disease, and eosinophilic granuloma). Histiocytosis-X is characterized by granulomatous formation with histiocytic infiltration and proliferation. The clinical manifestations in a given patient relate to the specific involved sites of disease and can range from a solitary eosinophilic granuloma in the medullary cavity of bone to acute disseminated fulminant disease.
  • a therapeutic composition containing A2 in saline is used to treat patients suffering from histiocytosis-X, as follows. All patients are skin tested prior to the first treatment. If there is no hypersensitivity reaction, the composition is administered intramuscularly, as described above. As long as continuing improvement is seen, treatment is continued until a remission occurs. If after 42 days there is no improvement in the patient's clinical condition, treatment is discontinued.
  • composition is effective to bring about remission in many patients.
  • Response varies with the clinical stage of the disease; patients in Stages I, II, and III respond better than patients in Stage IV.
  • H2R+ cells The clinical response of histiocytosis-X suggests a model for the disease and for the action of the therapeutic compositions of the invention.
  • the suppressor T-cells induced by the compositions of the invention are referred to as H2R+ cells; this terminology will now be further explained.
  • Histamine receptors are divided pharmacologically into two classes: H1 receptors are blocked by diphenhydramine-like antagonists, while H2 receptors are blocked by cimetidine-like agents. Physiologically, most effects of histamine are mediated by one or the other of these two receptors. For example, HI receptors modulate bronchoconstriction and ileal smooth muscle contraction, while H2 receptors mediate gastric secretion. H1 and H2 receptors are found on lymphocyte surfaces as well; HI receptors are found on approximately 35% of human peripheral blood T-cells, while H2 receptors are found on about 25% of peripheral blood T-cells.
  • H1 receptor bearing T-cells H1R+ cells
  • H2 receptor bearing T cells H2R+ cells
  • H1R+ T-cells are helper cells
  • H2R+ T-cells are suppressor cells. Disturbances in the expression and distribution of H1R+ helper cells and H2R+ suppressor cells have been associated with a variety of pathologic conditions.
  • H1R+ helper cell deficiency is described in patients with primary amyloidosis; excessive numbers of H2R+ suppressor cells are seen in malignancy; and a deficiency of H2R+ suppressor cells has been reported in patients with atopy/allergy and, as discussed above, in histiocytosis-X.
  • H2R+ suppressor T-cells are observed in many histiocytosis-X patients. Following treatment as described above, many of these patients no longer exhibit low H2R+ numbers. In addition, there is a significant correlation between clinical response and the ability of the compositions of the invention to induce. in vitro, H2 receptors on patient T-cells.
  • This correlation forms the basis for a simple in vitro screening test to select patients who are likely to respond to treatment.
  • the correlation also forms the basis for the following hypothesis for histiocytosis-X.
  • the immune system in health maintains a delicate balance between autoreactive and suppressor lymphocyte populations. When histiocytosis-X is present, there is an imbalance between these populations due to a relative deficiency of H2R+ suppressor T-cells.
  • the immunologic autoreactivity that results produces the clinical manifestations of the disease.
  • Successful treatment with the compositions of the invention restores the number of H2R+ cells to normal, resulting in the return of correct immune balance and consequence abrogation of clinical disease. Diagnostic Use
  • A2 and B1 will permit the production of polyclonal and monoclonal antibodies to A2 and B1 which can be labeled, e.g., radioactively or enzymatically, for use in a standard immunoassay to measure A2 or B1 in a clinical sample, e.g., blood, urine, or biopsied tissue.
  • A2 and B1 can, of course, be labeled, instead of the antibody.
  • Such assays will be useful in establishing causality in diseases whose relationship to immune system dysfunction is unclear, and will also facilitate diagnosis of difficult to diagnose syndromes such as histiocytosis-X; it is expected that histiocytosis-X patients and patients with other diseases characterized by suppressor T-cell deficiency will exhibit decreased A2 or B1 levels, compared to normal people.
  • B1 is a small peptide which, now that it has been isolated in pure form, will be able to be made using standard peptide synthesis techniques.
  • A2 now that it exists in pure form, will also be able to be made synthetically. Since A2 is too large to be made easily by existing peptide synthesis techniques, recombinant DNA technology will probably be used for large scale production.
  • the DNA sequence encoding A2 can be synthesized and inserted into a cloning vector, or a synthetic probe can be used to obtain A2-encoding mRNA, which will be used to produce the corresponding cDNA.
  • compositions containing any percentage of A2 greater than about 20%, w/w, can be used therapeutically.

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EP84902121A 1983-04-25 1984-04-25 Zusammensetzung zur induktion von suppressor-t-zellen und verfahren Withdrawn EP0140968A1 (de)

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US48794883A 1983-04-25 1983-04-25
US487948 2000-01-19

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SE448605B (sv) * 1980-10-27 1987-03-09 Vtoroi Mo G Med I Imen Terapeutiskt preparat for reglering av t-immunsystemet och forfarande for framstellning derav

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