EP0413721A1 - Human gm-csf variants - Google Patents
Human gm-csf variantsInfo
- Publication number
- EP0413721A1 EP0413721A1 EP89905045A EP89905045A EP0413721A1 EP 0413721 A1 EP0413721 A1 EP 0413721A1 EP 89905045 A EP89905045 A EP 89905045A EP 89905045 A EP89905045 A EP 89905045A EP 0413721 A1 EP0413721 A1 EP 0413721A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- csf
- human
- ala
- cells
- amino acid
- 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- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the production of variants or mutants of human granulocyte-macrophage colony-stimulating factor which have new and useful properties.
- Human granulocyte-macrophage colony-stimulating factor (h GM-CSF) is a glycoprotein of 19,O00MW produced by a number of normal and transformed cells. The gene coding for this molecule has been cloned and the cDNA used to transfect eukaryotic and prokaryotic cells to produce recombinant h GM-CSF (rh GM-CSF) (1).
- GM-CSF The range of actions of GM-CSF extends over the whole lineage of neutrophils, eosinophils and monocytes. Specifically GM-CSF stimulates the progenitors of these cells to proliferate and differentiate to become mature cells (2). In addition it stimulates mature cells to greater function. The stimulation of mature cells results in greater capacity to phagocytose and kill micro-organisms, kill antibody-coated tumor cells and generate superoxide anions (0 2 ⁇ ) in response to various stimuli (3). The purpose of this activation is presumed to enable the mature cells become better effector cells in inflammatory reactions. Therapeutically, the main indications for GM-CSF are for its effects on progenitor cells or mature cells.
- GM-CSF Using its effects on progenitor cells, GM-CSF is used in the treatment of bone marrow failure as seen in aplastic anaemia on chemotherapy or AIDS-induced marrow suppression. Although found to be an excellent therapeutic agent, some toxicity is associated and is mainly due to stimulation of mature cells causing blood vessel damage or thrombosis. The eosinophilia caused by GM-CSF appears especially damaging in this regard.
- the capacity to stimulate mature cells is especially relevant, and local as well as systemic application of GM-CSF can be envisioned.
- the capacity of GM-CSF-activated neutrophils and eosinophils -to kill tumor cells that have bound antibody is especially remarkable and could be used in tumor therapy.
- GM-CSF GM-CSF stimulates their growth (4,5).
- the administration of GM-CSF in these cases might stimulate tumor growth.
- mutant molecules with selectivity for either mature cells or progenitor cells would be useful and have their specific clinical indications.
- some mutants may be able to stimulate differentiation without growth, and may also be therapeutically useful in malignancies.
- site-directed mutagenesis reactions have been performed on the GM-CSF molecule to generate variants or mutants with desired therapeutic properties.
- This work took into account previous observations of differences between mature and immature myeloid cells in their interaction with h GM-CSF, namely differences in dose-response, signalling pathways and binding characteristics (6) .
- lack of amino acids 1-24 resulted in the complete loss of GM-CSF activities and absence of amino acids 1-18 greatly decreased the effect of GM-CSF with no dissociation of activities.
- the site-directed mutagenesis approach allowed the manufacture of homogenous GM-CSF analogs and a more precise study of the 14-24 region.
- a human GM-CSF variant or mutant characterised in that amino acid 20 (gin) and/or amino acid 21 (glu) of human GM-CSF is/are replaced by another amino acid.
- the substitute amino acid for one or both of amino acids 20 and 21 is alanine (ala).
- the present invention includes the following preferred mutant hGM-CSFs: h GM-CSF-ala 20 h GM-CSF-ala 21 h GM-CSF-ala 20 ' 21 In work leading to the present invention it has been established that:
- amino acids at position 20 and 21 are essential for h GM-CSF activity (their deletion results in complete loss of biological activity);
- a pharmaceutical composition comprising a human GM-CSF variant or mutant as described above.
- the use of such a composition may avoid some of the side effects of GM-CSF or provide new properties including the ability to block leukemic growth.
- substitutions of glu at position 21 and both gin at 20 and glu at 21 will selectively result in stimulation of mature cells. This may be partly due to the different affinity of GM-CSF receptors on mature cells and is reflected in the intermediate capacity of these mutants to inhibit binding of rh GM-CSF. This type of molecule will have special use in all cases when patients bear tumors whose stimulation should be avoided, and also in cases where control of the infectious process is of primary importance. Further substitution of amino acids 20 or 21 may alter the molecule to block function or to change the spectrum of action of the molecule. Blocking mutants will have use in malignancies that depend on GM-CSF for growth, and may be excellent agents to be used as adjuncts in chemotherapy.
- a human GM-CSF variant or mutant characterised in that a substantial number of the amino acids of human GM-CSF have been deleted with retention of maximal stimulation of mature cell function. Since amino acids 20 and 21, or replacement mutants thereof, have been-established as critical for GM-CSF function, it is preferred that the deletions in accordance with this invention do not include amino acids 20 or 21. In one embodiment of the invention described in greater detail below, the deletion of amino acids 14-18 of h GM-CSF has been found to be possible with retention of maximal stimulation of mature myeloid function.
- a pharmaceutical composition comprising a human GM-CSF "deletion" variant or mutant as described above.
- mutagenic primers were synthesized on an Applied Biosystems 381A DNA synthesizer, acrylamide gel-purified and 5' phosphorylated using T4 polynucleotide kinase.
- the DNA template for mutagenesis was mpl9GM, a bacteriophage carrying the GM-CSF cDNA.
- Oligonucleotide- primed site-directed mutagenesis was performed as described (7) . Briefly, the kinased mutagenic primer and M13 universal sequencing primer were hybridized to the single-stranded DNA template.
- the product was then extended and ligated, using the Klenow fragment of DNA polymerase and T4 ligase, for 16-20hrs at room temperature. Part of the resulting mixture was used to transform competent JM101 cells. The mutagenic primer was then 5* 32 P-labelled and used as a probe to identify plaques with the desired mutation. Positive clones were analysed by DNA sequencing using the chain termination method (8) to confirm the presence of the mutation.
- the mutated GM-CSF cDNA sequences were cloned into a ⁇ JL4 expression vector and used for transfection of COS cells by electroporation. After transient expression, the COS cell supernatants containing mutant GM-CSF protein were analysed for GM-CSF activity.
- GM-CSF sequences were cloned into a pAL181 vector and transformed into E.coli GI586. Mutant GM-CSF was expressed in these clones following temperature induction and released from the cells by disruption. Crude cell lysates were analysed for GM-CSF activity.
- Monocytes were obtained from the peripheral blood of normal donors after density gradient centrifugation on lymphoprep (Nyegaard Oslo, Norway). The monocytes were further purified by countercurrent elutriation in a Beckman JE-6B elutriator using a Sanderson chamber as described (11). Cytocentrifuge preparations were stained with Giemsa and only preparations judged to be 90% monocytes were used. Antibodv-deoendent cell-mediated cvtotoxicity assay (ADCC) .
- ADCC Antibodv-deoendent cell-mediated cvtotoxicity assay
- TNP trinitrophenyl
- Monocyte adherence to plastic was measured by an isotopic method. After labelling with 51 Cr, 3 x 10 5 cells were plated into microtitre wells either with or without the addition of stimulators in a total volume of lOO ⁇ l. The cells were incubated at 37°C 5% C0 2 for 16h. Following incubation in the microtitre wells, aliquots of supernatants were counted to provide a measure of spontaneous ⁇ Cr release. Remaining supernatants were then removed by aspiration and the cells washed three times to remove non-attached monocytes. The cells were lysed by the addition of lysis buffer containing lOmM tris-HCl, 0.15M NaCl and 1% NP40.
- competition experiments were performed with lOOpg - 2 -I-rh GM-CSF and 4 x 10- neutrophils in 0.15ml incubated at 10°C for 3hr. Cell bound radiolabel was determined by centrifuging through fetal calf serum.
- the stimulation of human bone marrow cells with rh GM-CSF resulted in the formation of colonies after 14 days of culture.
- the full length GM-CSF (1-127) was as active as the recombinant molecule, and the mutants with deletions of amino acids 1-24, 7-24, 14-24 and 20-21 were inactive (Table I).
- the mutant gin 20 ala stimulated maximal number of colonies similar to the full length GM-CSF. From dose response curves this mutant appeared to be more powerful than the parent molecule, but at this stage activity per mg of protein is not known.
- the other mutants stimulated submaximal number of colonies in the order: deletion 14-18; substitution glu 21 to ala; substitution gin 20 and glu 21 to ala, ala. Stimulation of human granulocyte function.
- Granulocyte function was stimulated by the recombinant h GM-CSF as measured in the ADCC and superoxide anion release (Table I) assays. Similarly to the proliferation experiments, the full length (1-127) GM-CSF was active while the mutants with deletion of amino acids 1-24, 7-24, 14-24 and 20-21 were inactive. In startling contrast, however, maximal stimulation could be obtained with the three mutants, deletion 14-18, substitution glu 21 to ala, and substitution gin 20, glu 21 to ala, ala, all of which stimulated submaximal number of colonies in the proliferation/differentiation assay. The mutant substitution gin 20 to ala stimulated maximal activity, like the full length 1-127 GM-CSF.
- GM-CSF mediated function (% maximum-) Stimulus Day 14 colony ADCC b FMLP stimulated Displace- formation 0 2 ⁇ production 0 ment of rh GM-CSF binding"
- Table II shows the results of tests performed with GM-CSF (1-127) and the mutant with substitution of glu 21 for ala produced by different methods. It will be seen that the mutant ala 21 produced by either of the three methods described here does not maximally stimulate day 14 colony formation although it can maximally stimulate neutrophil and monocyte function.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
L'invention concerne une variante ou un mutant de facteur de croissance des colonies de granulocyte-macrophages (GM-CSF), caractérisé en ce que l'amino acide 20 (gln) et/ou l'amino acide 21 (glu) de GM-CSF humain est/sont remplacés par un autre amino acide, et alternativement ou additionnellement, une quantité substantielle des amino acides de GM-CSF humain sont éliminés.The invention relates to a variant or mutant of granulocyte-macrophage colony growth factor (GM-CSF), characterized in that the amino acid 20 (gln) and / or the amino acid 21 (glu) of GM -CSF human is / are replaced by another amino acid, and alternatively or additionally, a substantial amount of the amino acids of human GM-CSF are eliminated.
Description
Claims
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPI784888 | 1988-04-21 | ||
AU7848/88 | 1988-04-21 | ||
AU9943/88 | 1988-08-19 | ||
AUPI994388 | 1988-08-19 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0413721A1 true EP0413721A1 (en) | 1991-02-27 |
EP0413721A4 EP0413721A4 (en) | 1991-11-13 |
Family
ID=25643468
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19890905045 Withdrawn EP0413721A4 (en) | 1988-04-21 | 1989-04-21 | Human gm-csf variants |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0413721A4 (en) |
JP (1) | JPH03503897A (en) |
AU (1) | AU615550B2 (en) |
WO (1) | WO1989010403A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2060699A1 (en) * | 1991-02-11 | 1992-08-12 | Isia Bursuker | Gm-csf inhibiting oligopeptides |
JPH09501154A (en) * | 1993-07-28 | 1997-02-04 | メドベット サイエンス プロプライアタリー リミティド | Hematopoietic growth factor antagonist |
US6465616B1 (en) | 1994-04-08 | 2002-10-15 | Bresagen Limited | Interleukin-5 antagonist |
AUPN378095A0 (en) | 1995-06-23 | 1995-07-20 | Bresagen Limited | Haemopoietic growth factor antagonists and uses therefor |
AU703052B2 (en) * | 1995-06-23 | 1999-03-11 | Bresagen Limited | Haemopoietic growth factor antagonists and uses therefor |
WO2018227142A1 (en) * | 2017-06-09 | 2018-12-13 | The Regents Of The University Of Colorado, A Body Corporate | Gm-csf mimetics and methods of making and using same |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU594014B2 (en) * | 1984-03-21 | 1990-03-01 | Research Corporation Technologies, Inc. | Recombinant DNA molecules |
AU588819B2 (en) * | 1984-10-29 | 1989-09-28 | Immunex Corporation | Cloning of human granulocyte-macrophage colony stimulating factor gene |
CN1031465C (en) * | 1984-11-20 | 1996-04-03 | 先灵生物技术公司 | The peptide cDNA clone that existing human granulocyte meloschisis phagocyte of coding schedule and eosinophie cellular growth activity are many |
US5391485A (en) * | 1985-08-06 | 1995-02-21 | Immunex Corporation | DNAs encoding analog GM-CSF molecules displaying resistance to proteases which cleave at adjacent dibasic residues |
JPS63502795A (en) * | 1985-10-03 | 1988-10-20 | バイオジェン インコーポレイテッド | Granulocyte-macrophage colony-stimulating factor-like polypeptides, DNA sequences, recombinant DNA molecules and methods for producing high yields of human granulocyte-macrophage colony-stimulating factor-like polypeptides in microbial cells. |
DE3545568A1 (en) * | 1985-12-21 | 1987-07-16 | Hoechst Ag | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
AU1369088A (en) * | 1987-02-06 | 1988-08-24 | Genetics Institute Inc. | Colony stimulating factors having reduced levels of carbohydrate |
WO1989000579A1 (en) * | 1987-07-16 | 1989-01-26 | Schering Corporation | Purification of gm-csf |
AU626530B2 (en) * | 1987-07-17 | 1992-08-06 | Schering Biotech Corporation | Human granulocyte-macrophage colony stimulating factor and muteins thereof |
-
1989
- 1989-04-21 EP EP19890905045 patent/EP0413721A4/en not_active Withdrawn
- 1989-04-21 AU AU35494/89A patent/AU615550B2/en not_active Ceased
- 1989-04-21 JP JP1504852A patent/JPH03503897A/en active Pending
- 1989-04-21 WO PCT/AU1989/000177 patent/WO1989010403A1/en not_active Application Discontinuation
Non-Patent Citations (3)
Title |
---|
Journal of Immunology, Vol. 141, No. 3, August 1, 1988, USA, pages 881-889; IAN CLARK-LEWIS et al.: "Structure-functions studies of human granulocyte-macrophage colony stimulating factor", Abstract; figures 2,5,6; table II. * |
Lymphokine Research, Vol. 6, No. 1, 1987, New York, USA, A.F. LOPEZ et al.: "Biological properties of recombinant and chemically-synthesized human granulocyte-macrophage colony-stimulating-factor(GM-CSF)", Abstract No. 1322. * |
See also references of WO8910403A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1989010403A1 (en) | 1989-11-02 |
AU3549489A (en) | 1989-11-24 |
EP0413721A4 (en) | 1991-11-13 |
JPH03503897A (en) | 1991-08-29 |
AU615550B2 (en) | 1991-10-03 |
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