AU615550B2 - Human gm-csf variants - Google Patents

Description

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0- g OPI DATE 2 4 /11/89 6 1 5 AOJP DATE 21/12/89 APPLN- ID 94 89 PCT NUMBER PCT/AU89/00177 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 International Publication Number: WO 89/10403 C12N 15/00, C2P 21/02 Al C7K 15/00, A6IK 37/02A (43) International Publication Date: 2 November 1989 (02.11.89) (21) International Application Number: PCT/AU89/00177 (74) Agents: SLATTERY, John, Michael et al.; Davies Collison, I Little Collins Street, Melbourne, VIC 3000 (AU).

(22) International Filing Date: 21 April 1989 (21.04.89) (81) Designated States: AT (European patent), AU, BE (Euro- Priority data: pean patent), CH (European patent), DE (European pa- PI 7848 21 April 1988 (21.04.88) AU tent), FR (European patent), GB (European patent), IT PI 9943 19 August 1988 (19.08.88) AU (European patent), JP, LU (European patent), NL (European patent), SE (European patent), US.

(71) Applicant (for all designated States except US): MEDVET SCIENCE PTY. LTD. [AU/AU]; Frome Road, Ade- Published laide, S.A. 5000 With international search report.

(72) Inventors; and Inventors/Applicants (for US only) VADAS, Mathew, Alexander [AU/AU]; 8 Branch Road, Stirling, S.A. 5152 LOPEZ, Angel, Francisco [AR/AU]; 142 Stanly Street, North Adelaide, S.A. 5006 SHANNON, Mary, Frances [AU/AU]; 8 The Crescent, Crafers, S.A.

5152 (AU).

(54) Title: HUMAN GM-CSF VARIANTS 1 10 20 A PARSPSTQPWEHVNATQEAR RLLNLSR 54 C C 121 127--- C alanine (ala) phenylalanine arginine serine threonine glutamine (gin) trytophan glutamic acid (glu) histidine valine N asparagine I isoeucine L leucine C cysteinc (57) Abstract A human granulocyte-macrophage colony-stimulating factor (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, and alternatively or additionally, a substantial number of the amino acids of human GM-CSF are deleted.

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I t WO 89/10403 PCT/AU89/00177 'HUMAN GM-CSF VARIANTS 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 19,000MW 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) 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 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 in response to various stimuli The purpose of this activation is presumed to enable the mature cells become better effector cells in inflammatory reactions.

WO 89/10403 PCT/AU89/00177 Therapeutically, the main indications for GM-CSF are for its effects on progenitor cells or mature cells.

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.

In the treatment of infections, 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.

It should be noted that certain tumors have receptors for GM-CSF and GM-CSF stimulates their growth The administration of GM-CSF in these cases might stimulate tumor growth. Thus, mutant molecules with selectivity for either mature cells or progenitor cells would be useful and have their specific clinical indications. In addition, some mutants may be able to stimulate differentiation without growth, and may also be therapeutically useful in malignancies.

Based on these considerations, site-directed mutagenesis reactions have been performed on the GM-CSF i 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 With that approach, lack T WO 89/10403 PCT/AU89/00177 3 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. This allowed establishment of amino acids 20 and 21 as critical for GM-CSF function.

Accordingly, in a first aspect of the present invention, there is provided a human GM-CSF variant or mutant, characterised in that amino acid 20 (gln) and/or amino acid 21 (glu) of human GM-CSF is/are replaced by another amino acid.

Preferably, the substitute amino acid for one or both of amino acids 20 and 21 is alanine (ala).

Accordingly, the present invention includes the following preferred mutant hGM-CSFs: h GM-CSF-ala 20 h GM-CSF-ala 2 1 h GM-CSF-ala 20 21 In work leading to the present invention it has been established that: 1. amino acids at position 20 and 21 are essential for h GM-CSF activity (their deletion results in complete loss of biological activity); and 2. substitutions of amino acids at positions 20 and 21 lead to alterations in the potency of the h GM-CSF molecule and to dissociation between proliferation/differentiation of progenitor cells and functional activation of mature cells. In particular, proliferation and differentiation of myeloid cells is greatly reduced while maximal stimulation of mature cells is still achieved.

~I I 1 1 WO 89/10403 PCT/AU89/00177 4 i Accordingly, in a further aspect of the present invention, there is provided 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.

As demonstrated with GM-CSF-ala 2 0 substitution of amino acid gln at this position may replace GM-CSF as a more potent therapeutic molecule. Clearly higher doses of 1j* 10 any therapeutic agent are not desirable especially when the subcutaneous route appears to be the preferred way of administering this agent.

As demonstrated with GM-CSF-ala 21 and GM-CSF-ala 20 21 substitutions of glu at position 21 and both gln 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 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.

In addition to the above "substitution" variants or mutants, it has also been established that a mutant h GM-CSF with deletion of certain amino acids stimulated submaximal number of colonies after 14 days when tested i for stimulation of human bone marrow cells in a iV proliferation/differentiation assay but that maximal I ii 1 L._ii-J WO89/10403 PCT/AU89/00177 stimulation of granulocyte function could be obtained with this deletion mutant as measured in ADCC and superoxide anion release assays.

Accordingly, in accordance with another aspect of the present invention there is provided 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.

Accordingly, in a further aspect of the present invention, there is also provided a pharmaceutical composition comprising a human GM-CSF "deletion" variant or mutant as described above.

Further details of the present invention, including the production of the human GM-CSF variants or mutants by site-directed mutagenesis and expression in COS cells or Escherichia coli, are illustrated by way of example in the following detailed description. In addition, the following description shows that a chemically synthesized GM-CSF peptide containing ala 21 has similar properties to the mutant containing this substitution made by sitedirected mutagenesis.

EXAMPLE 1 MATERIALS AND METHODS Oliaonucleotide directed mutaaenesis Synthetic mutagenic primers were synthesized on an Applied Biosystems 381A DNA synthesizer, acrylamide i hI lJ- .1 ~7~sma~ WO 89/10403 PCT/AU89!06177 gel-purified and 5' phosphorylated using T4 polynucleotide kinase. The DNA template for mutagenesis was mpl9GM, a bacteriophage carrying the GM-CSF cDNA. Oligonucleotideprimed site-directed mutagenesis was performed as described 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 to confirm the presence of the mutation.

The mutated GM-CSF cDNA sequences were cloned into a pJL4 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.

Certain of the mutated 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.

A GM-CSF peptide containing ala 21 was chemically synthesized by Dr. Ian Clark-Lewis (Biomedical Research Centre, University of British Columbia, Vancouver, Canada) on behalf of the inventors, using automated stepwise solid-phase methods as described 4.

k 1' V :t WO 89/10403 PCT/AU89/00177 7 Clonal Proliferation assays: Bone marrow cultures.

Myeloid colony-forming unit assay.

Light density non-adherent bone marrow cells were obtained by separation on a Ficoll-Paque (Pharmacia, Sweden) density gradient followed by monocyte depletion using carbonyl iron. Cells were cultured in 0.3% agar (Difco, with Iscove's Modified Dulbecco's Medium (Commonwealth Serum Laboratories, Australia), 30% fetal calf serum (FCS) (Gibco, 0.66% bovine serum albumin (Fraction V, Sigma, and mercaptoethanol 2 x 10- 5 M at a concentration of 5 x 10 4 cells/l ml culture and rh GM-CSF (100ng/ml) or appropriate mutant GM-CSF proteins.

Aggregates of more than 40 cells were scored as colonies after 14 days incubation. The agar discs were then fixed with 3% glutaraldehyde and transferred onto individual 5 x 8cm glass slides. The discs were dried at room temperature and stained with luxol fast blue and a combined specific and non-specific esterase stain.

Mature cell function assays: Purification of human neutrophils, eosinoohils and monocytes.

Peripheral blood of healthy volunteers was centrifuged on a hypertonic gradient of Metrizamide (Nyegaard, A/C, Oslo) as previously described (10) after dextran sedimentation. The purity was greater than for neutrophils and greater than 92% for eosinophils. The cells were resuspended in Eagle's Minimal Essential Medium supplemented with 10% FCS, 20mM HEPES buffer, and antibiotics.

Monocytes were obtained from the peripheral blood of normal donors after density gradient centrifugation on Ir 1A WO 89/10403 PCT/AU89/00177 8 lymphoprep (Nyegaard Oslo, Norway). The monocytes were further purified by countercurrent elutriation in a Beckman JE-6B elutriator using a Sanderson chamber as described Cytocentrifuge preparations were stained with Giemsa and only preparations judged to be monocytes were used.

Antib.ody-deendent cell-mediated cvtotoxicity assay (ADCC).

of 51 Cr-labelled, trinitrophenyl (TNP)-coupled P815 cells (4 x 103) were incubated with 24pl of rabbit IgG anti-TNP (Miles-Yeda, Rehovot, Israel), 80.l of purified human neutrophils or eosinophils (1.3 x 105) as effector cells, and 16il of rh GM-CSF for 2.5h at 37 0 C in V-bottom microtitre plates. Percent cytotoxicity was calculated as described previously (12).

Superoxide production.

Purified neutrophils or eosinophils were incubated with medium or with gIL-3, rh GM-CSF or medium for various times at 37 0 C. 150pl of cells were then added to a mixture of l00pl freshly prepared cytochrome C (Sigma, type VI, 12.4 mg/ml), 100pl of FMLP (Sigma) and made up to Iml with medium. The mixtures were incubated at 37 0 C for min, after which the cells were rapidly cooled, pelleted at 4 0 C and the supernatants transferred to plastic disposable cuvettes. Superoxide production was measured in duplicate by the reduction of cytochrome C as described In each experiment superoxide dismutase (Sigma, St.Louis, MO) inhibited all 02- generation.

Measurement of monocyte adherence.

Monocyte adherence to plastic was measured by an isotopic method. After labelling with 51 Cr, 3 x 105 cells were plated into microtitre wells either with or without the addition of stimulators in a total volume of 100pl.

The cells were incubated at 37C 5% CO 2 for 16h.

Following incubation in the microtitre wells, aliquots of WO 89/10403 PCT/AU89/00177 9 supernatants were counted to provide a measure of spontaneous 51 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 tris-HCl, 0.15M NaC1 and 1% NP40. The contents of each well was then counted in a Packard gamma counter. The percent adherence was determined: Adherence 51 Cr cpm in lysate/total cell associated 51 Cr cpm added x 100 where total cell associated 51 Cr cpm is calculated as the total 51 Cr cpm added minus the 51Cr cpm spontaneously released.

Binding of 12-I-rh GM-CSF to neutrophils.

rh GM-CSF was radioiodinated by the two-phase method with no detectable change in biologic activity. 1 25 I-rh GM-CSF specifically bound to human neutrophils with a single affinity of sites with apparent KD 7+1 x 10-11M and 140+50 molecules bound per cell (mean SEM, m For assessment of binding by mutant GM-CSF proteins, competition experiments were performed with 100pg 125 I-rh GM-CSF and 4 x 106 neutrophils in 0.15ml incubated at for 3hr. Cell bound radiolabel was determined by centrifuging through fetal calf serum.

RESULTS

Clonal proliferation and differentiation of myeloid cells.

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 The mutant gin 20 ala stimulated maximal number of colonies similar to the full length i WO 89/10403 PCT/AU89/00177 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 gln 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 gln 20 to ala stimulated maximal activity, like the full length 1-127 GM-CSF.

TABLE I Properties of GM-CSF mutants GM-CSF mediated function maximuma) Stimulus Day 14 colony ADCCb FMLP stimulated Displaceformation 02- productionc iment of rh GM-CSF bindingd Nil 0 0 0 0 rh GM-CSF 100 100 )9 100 100 1-127 9 9 6 e 100 7 0 t 4 5 h 613 A 1-24 6±6 9 9 h 0 16±2 3 i i WO 89/10403 PCT/AU89/00177 11 A 7-24 0 0 23±23 14±1 A 14-18 32±13 80±9 88±2 27±7 A 14-24 6±1 0 14±14 ND gln 20 ala 101±32 114±1 80±10 62±3 glu 21 ala 15±8 86±18 67±12 29±9 gln 20 glu 21 ala,ala 4±2 80±1 63±19 15±3 A 20,21 0±0 0 7±7 13±1 COS cell supernatant f 2±2 0 0 0 Note: A indicates deletion.

In order to compare experiments, results are normalised to plateau level of rh GM-CSF.

Antibody-dependent cell-mediated cytotoxicity.

30 min preincubation at 37 0 C before stimulation with 7 M f-met-leu-phe (FMLP).

Competition for binding to neutrophils between a 1/2 dilution of supernatant and 100pg 1 25 I-rh GM-CSF.

Arithmetic mean SEM of three experiments performed on different bone marrow donors.

Supernatant from untransfected COS cells.

Untransfected COS cells contained some capacity to activate in this assay. Comparisons are therefore between 1-127 (maximum) and COS cell supernatant (minimum).

Arithmetic mean of two experiments each performed in triplicate.

EXAMPLE 2 MATERIALS AND METHODS As in Example 1 above.

-140 WO 89/10403 PCT/AU89/00177 12

RESULTS

Table II shows the resultF of tests performed with GM-CSF (1-127) and the mutant with substitution of glu 2 1 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.

TABLE II Properties of GM-CSF ala 2 produced by different methods GM-CSF mediated function maximuml- FMLP- Displace- Monocyte Stimulus Day 14 ADCCb stimulated ment of rh adherencee 02- pro- GM-CSF ductionc bindingd Nil O O 0 O 0 rh GM-CSF 100 10 0)9 100 100 100 Site-directed mutagenesis and expression in: COS cells 1-127 9 9 6 f 100 70 45 h 61±3 100 ala 2 1 15±8 86±18 67±12 29±9 84±10 E.coli 1-127 138±40 ND 76±12 ND ND ala 21 23±7 ND 71±10 ND ND Chemical 1-127 163±48 94±5 ND 100 100 synthesis ala 21 28±16 91±4 ND 74 80±9 1 WO 89/10403 PCT/AU89/00177 13 In order to compare experiments, results are normalised to plateau level of rh GM-CSF.

Antibody-dependent cell-mediated cytotoxicity.

30 min preincubation at 37 0 C before stimulation with 10- 7 M f-met-leu-phe (FMLP).

Competition for binding 100pg 125 I-rh GM-CSF to neutrophils.

Purified human monocytes were incubated for 16h at 37 0 C in microtitre plates and the percent adherent monocytes calculated after washing three times.

Arithmetic mean SEM of three experiments performed on different bone marrow donors.

Untransfected COS cells contained some capacity to activate in this assay. Comparisons are therefore between 1-127 (maximum) and COS cell supernatant (minimum).

In addition a substitution of glu 21 for arg increased monocyte adherence by 42.4% indicating that the replacement of glu at 21 not only by ala but also by another residue alters the activity of the GM-CSF molecule.

3 r .L~Ili i L Li WO 89/10403 PCr/AU89/00177 14 1. Wong, J.S.'Witek, P.A.Ternple, K.M.Wilkens, A.C.Leary, D.P.Luxenberg, S.S.Jones, E.L.Brown, R.M.Kay, E.C.Orr, C.Shoemaker, D.W.Golde, R.J.Kaufrnan, R.M.Hewick, E.A.Wang, and S.C.Clark.

(1985) Science (Wash.DC). 221:810-815.

2. Metcalf, C.G.Begley, G.R.Johnson, N.A.Nicola, M.A.Vadas, A.F.Lopez, D.J.Williamson, G.G.Wong, S.C.Clark, and E.A.Wang. (1986). Blod £L-:37-45.

3. Lopez, D.J.Williamson, J.R.Garnble, C.G .Begley, J.M.Harlan, S. J.Klebanoff, A.Waltersdorph, G.Wong, S.C.Clark, and M.A.Vadas.

4. Dedhard, Gaboury, Galloway, P. and Eaves, C. Proc.Natl.Acad.Sci.USA B5-:9253-9257, (1988).

Berdel, Danhauser-Riedi, Steinhauser, G.

and Winton, E.F. Blo 231:80-83, (1989).

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Claims (6)

1. A human granulocyte-macrophage colony stimulating factor (GM-CSF) variant or mutant, characterised in that amino acid 20 (gln) and/or amino acid 21 (glu) of human GM-CSF is/are replaced by another amino acid, and alternatively or additionally, amino acids 14 (glu) to 18 (ala) of human GM- CSF are deleted.

2. A human GM-CSF variant or mutant according to claim 1, wherein the replacement amino acid for one or both of amino acids 20 and 21 of human GM-CSF is alanine (ala).

3. Human GM-CSF-ala

4. Human GM-CSF-ala 2 1 Human GM-CSF-ala 20 2 1 S6. A human GM-CSF variant or mutant according to claim 1, wherein amino acids 14-18 are deleted.

7. A pharmaceutical composition comprising a human GM-CSF variant or mutant according to any of claims 1 to 6, in association with one or more pharmaceutically acceptable carriers or diluents.

8. A method of stimulation of human granulocyte function, which comprises administration of a stimulation effective amount of a human GM- CSF variant or mutant according to any of claims 1 to 6. Dated this 2nd day of May, 1991. MEDVET SCIENCE PTY. LTD. By its Patent Attorneys DAVIES COLLISON 910502jmscmc.00 8,35494 et,16 7 0 7