AU2007200889A1 - Gradual modification super-agonists and antagonists of signal-proteins and peptides - Google Patents
Gradual modification super-agonists and antagonists of signal-proteins and peptides Download PDFInfo
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- AU2007200889A1 AU2007200889A1 AU2007200889A AU2007200889A AU2007200889A1 AU 2007200889 A1 AU2007200889 A1 AU 2007200889A1 AU 2007200889 A AU2007200889 A AU 2007200889A AU 2007200889 A AU2007200889 A AU 2007200889A AU 2007200889 A1 AU2007200889 A1 AU 2007200889A1
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Description
28/02 2007 16:21 FAX COLLISON CO IP AUST CANBERRA l009/095 o 47392DIV2 DIA:JO SP/00/011 CRegulation 3.2
AUSTRALIA
C Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT 0o
ORIGINAL
00 oi 0 Name of Applicant: STICHTING PHARMA PARK IP Actual Inventors: VICTOR SMIT and WILLEM HUPPES Address for Service: COLLISON CO., 117 King William Street, Adelaide, S.A. 5000 Invention Title: GRADUAL MODIFICATION SUPER-AGONISTS AND ANTAGONISTS OF SIGNAL-PROTEINS AND PEPTIDES Divisional Application Details This is a Divisional of Australian Patent Application Nos.
32668/95 dated 30 August 1995 43499/99 dated 11 August 1999 2003248017 dated 18 September 2003 The following statement Is a full description of this invention, including the best method of performing it known to me/us: COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28102 2007 16:21 FAX COLLISON CO IP AUST CANBERRA 1i010/095 1A GRADUAL MODIFICATION, SUPER-AGONISTS AND ANTAGONISTS 0 OF SIGNAL PROTEINS AND PEPTIDES
C)
Scientific field of the invention: 00 c-I This invention is on the area of the chemical modification technology of biologically active proteins and peptides. More specifically it concerns use of chemical modification to obtain a protein or peptide with superior properties or with new or even counteracting 00 5 properties. In addition, the invention also concerns a new method for structure-function 00 0 analysis by using gradual chemical modification, a biological principle, namely the ^0 catalytic activity of signal peptides and the successful abrogation thereof, leading to a very effective inhibitor of Acute Myeloid Leukemia cells.
O As illustration, but not as limitation, we show the chemical modification of human IL-3, a protein that also has a substantial therapeutic value after modification. This patentdescription also contains specific examples within the therapeutic field of the invention.
Field of the anplications: There are two possible fields of applications that are important for the invention: A field for an IL-3 with superior properties (Superagonist) or a field for an IL-3 with counteracting or new properties (Antagonist). In this patent description Superagonists are for instance an IL-3 with a lowered antigenecity and/or a higher biological activity and/or a higher stability.
Possible ipplications of IL-3 Superagonists are: reduction of the cytopenic phase after myelo-ablative therapy like after inductive therapy for bone marrow transplantation or after accidental radiation.
induction of a synchronized cell-cycle of cells with an IL.3 receptor, for instance for chemotherapy of leukemia's.
induction of enhancement of the IL-3-dependent progeny both for the number of cells and their activation, for treatment of diseases like worm infections, tuberculosis, fungal infections and certain viral infections.
S selective outgrowth of the bone marrow towards nucleus containing cells except lymphocytes, for instance with burning wounds and non-homologous skintransplants, Some, but not all, examples of applications of signal-substance antagonists (with counteracting or cell-inhibitory activity), more specifically of IL-3 are: inhibition and/or neutralization of myeloid cells in bone marrow transplantation.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:21 FAX COLLISON CO IP AUST CANBERRA I011/095 2 0 o myelo-suppression in auto immune-diseases, cancer and diseases of the blood forming organs, like sickle cell anemia and thallisemia.
treatments to cure all sorts of cancer that involve cells with the IL-3 receptor, more T specifically almost all forms of acute'myeloid leukemia or chronic myeloid 0 5 leukemia, B-cell lymphoid tumors or other forms of cancer that are stimulated by IL-3, for instance certain follicle cell tumors.
induction of self-tolerance towards the tissues in auto immune diseases like 00 arthritis, rheumatic arthritis and diseases of the central nervous system, by o suppression or elimination of lymphoid cells that contain the IL-3 receptor. This can also lead to the impaired generation and elimination of effector cells like the eosinophilic granulocyte. Finally it is possible that there is a direct interaction with these cells, thus enabling a direct cure of the eosinophilic syndrome. This is CI also of great importance in acute phases of worm infections and hypersensitivity reactions to for instance medicinal drugs.
curing eosinophilic syndromes like eosinophilic gastritis and enteritis, fascitis, granulomatosis, sinusitis, pneumonia, asthma, Churg Strauss syndrome and other angitis treatments of the shock syndrome for instance by killing or suppressing the number of effector cells.
ablation or suppression of cells with an IL-3 receptor, like lymphoid cells and/or effector cells like the eosinophilic granulocyte, for the treatment of allergies. In these cases both suppression of the allergy and induction of tolerance towards the antigen are possible.
other allergic reactions where the action of IL-3 is involved.
treatments to prevent metastases that are stimulated by IL-3 mediated adhesion.
treatment of infectious diseases, for instance by suppressing an acute phase where there is an occurrence of excessive amounts of growth factor in the blood stream.
Treatment of HIV-infection and/or AIDS by suppression of B-cells and B-cell antibody production (that protect the HIV-virus against the cellular resistance of the host).
One or more of these applications can also be mentioned for other growth factors like the other Interleukins I-8 GM-CSF, TNF and gamma IFN, which are also good candidates for modification according to this invention. Hereby 'IL-3' is to be replaced by the other signal substance. Since the modes of interaction of the various substances in the various diseases can differ from substance to substance, several synergistic effects can also be expected. Therefore these are also ebodied in the invention. Finally, the following applications can be added or specified: COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:22 FAX COLLISON CO IP AUST CANBERRA W012/095 3
O
S- IL-1 inhibition'ro suppress IL-I stimulated metastases of melanoma's and forms of lung cancer.
IL-1 inhibition to suppress Alzheimer's disease S- IL-2 inhibition to suppress capillary leak syndrome 00 5 IL-2 inhibition to suppress periodontitis L-4 inhibition to suppress periodontitis IL-4 inhibition to suppress IL-4 stimulated viruses like for instance the radiation 00 leukemia virus in mice, 00 o IL-5 inhibition to suppress IL-5 stimulated respiratory tract infections.
S 10 IL-6 inhibition to suppress Rheumatoid arthritis.
IL-10 inhibition to suppress infections of Mycobacterium.
Cl With an in vivo example it is shown that there is a possibility to effectively suppress an AIDS-virus infection. This suppression is based on the reduction of the antibody-level that can be achieved by the suppression of antibody-producing B-cells. The fact that this can lead to an effective suppression of AIDS can be attributed to the following causes: (1) The HIV-virus infects the hystiocytic cells (macrophage like cells) preferably by opsonisation with antibodies. Macrophage tropism and the necessity to of infection of infection of these histiocytic cells for persisting infection is described in AIDS Res. and Human Retroviruses 9: 669 (1993) and references. By the reduction of the antibodylevels this form of infection can be suppressed. Antibodies can protect HIV andlor HIV-infected cells against cellular immunity. This explains the defective overall resistance in the asymtomatic phase of the HIV infection in spite of very high levels of in vitro neutralizing antibodies And a demonstrated cellular resistance. As demonstrated in the example the lowering of the antibody-levels can result in an effective cellular immunity against the virus and the virus infected cells and even the elimination of the virus.
Therefore, the inhibition of B-cells with for instance antagonists can lead to a cure of the HIV infection.
Since it is perceptible that the modified signal peptides can also be generated by molecular biology, these mutant proteins. DNA-constructs and the use thereof are also considered to be within the scope of the invention. Hereby, one can include 3ene-therapy with the constructs that contain the code for such peptides and/or proteins. The use in genetherapy can result in cells that are producing and excreting the super-agonist or the antagonist. Therefore, in one of the examples there is also an elaboration on the possibility to construct a growth factor with a reduced stability. This can be used especially in combination with antagonistic action, thus resulting in a selective administration whereby the high rate of breakdown of the antagonist can contain the COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:22 FAX COLLISON CO IP AUST CANBERRA 1a013/095 4 Saction to a very localized environment. This is particularly interesting in gene-therapy for .I solid tumors. If there is such a cell in or near the tumor, especially this tumor will Sexperience the exposure to the maximal effect. If there is an additional instability of the Sproduced signal peptide, the effect of the peptide can be very localized, thus leading to the 00 5 expectation of fewer side-reactions. In one of the examples it is demonstrated that it is possible to generate a signal peptide with a lowered stability. Since such peptides can also be generated with deletion and/or substitution mutants, such gene-therapeutic applications 0 and gene therapy- constructs are also considered to be within the field of applications of o the invention.
0 r- 10 This patent-description also elaborates on the use of chemical modification, protease Streatment and mass spectrometry. This can be applied to every modification of every N. peptide or protein, provided it can be specifically fragmented for instance with protcase's. The quantitative structure-function analysis part of the description contains the successful use of the combination of gradual chemical modification, protease treatment and laser desorption mass spectrometry with biological assays. Therefore, the application of this analysis on any peptide and/or protein that can be modified, specifically fragmented and is biologically active is also considered to be within the scope of the invention. It is also possible to use other mass spectrometric techniques like electrospray mass spectrometry, which is particularly suitable after for instance exoprotease treatment.
Finally, the description of the invention also discloses the importance of metal ions with more than 1 valence in charge, by preference Zinc-ions, especially for catalytic activity and the subsequent efficacy. Therefore, it is possible to influence the growth factor action by manipulating the (local) metal ions concentrations. Since there is a usual optimum in efficacy of the metal ions concentration, and any concentration below or beyond this optimum can lead to reduction of efficacy of the growth factor. This enables manipulation of the efficacy of the growth factor. In this way also indirect therapeutic effects can be achieved with metal-, preferably Zinc ions. This can be used in treatment of skin-diseases like wrath's in the form of ointments. Also, the use of inhalation-sprays for treatment of lung-affects is possible.
Backeround of the inventon: Human Interleukin-3: Interleukin-3. which was first isolated from T-cells as a glycoprotein, has been demonstrated to act on bone-marrow cells. Either with or without other growth factors it has been shown to induce the formation of various blood cells from these bone-marrow COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:22 FAX COLLISON CO IP AUST CANBERRA I014/095 O cells, Human IL-3 has been cloned for the first time in 1987 by Dorssers el al. who used Ci a humar. c-DNA library and hybridization with a probe of mouse DNA Gene 55: 115 C) (1987).
00 Structure-function relationship of human Interleukin-3: Several articles have been published concerning the structure-function relationship of human Interleukin-3 Biol. Chem. 266: 21310 (1991); J. Clin. Invest 90: 1879(1992); ON J. Immunol. 146: 83 (1991); EMBO J. 10:2125 (1991); Proc. Natl. Acad. Sci. USA 89: 00 11842(1992)).
0 o In their research they all used molecular biological techniques like the generation of deletion and substitution mutants. In the case that substitution mutants were used, the Schoice was based on the amino-acid homology oflL-3 with other species (mouse, rhesus monkey, gibbon) or it was based on deliberate changes of polarity or structure. With deletion mutants the protein was scanned for biological importance by removal of parts of the protein. Because of practical problems with regard to purification of the mutantproteins(muteins) these mutant proteins have never been checked for major structural changes. This is a major problem since these structural changes usually do occur. As described, sometimes they are even deliberately introduced. As a result any statement regarding the involvement of various amino acids on the biological activity can only be made with the greatest reservations.
Present invention has a different approach. The native IL-3 is used as starting material.
By using stepwise gradually increasing modification an extra selectivity is introduced in the chemical reaction. The modification that occurs simultaneous with the first change in biological activity can be simply localized with specific protease's and new forms of mass spectrometry. As a result, important amino acid residues can be quickly localized and minimal change and therefore maximal control) can be achieved in the introduction or change of a desired property. There is no need to purify the modified material and thus there are no problems in losses during purification's. This again enables an easy verification of the secondary structure. Therefore this invention is an improvement as compared to molecular biological techniques because the approach is more convenient, better verifiable and faster than the molecular biological approach.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16.-23 FAX COLLISON CO IP AUST CANBERRA I015/095 6 O Modified growth factors: Superagonists; In many structure-function research efforts on the various growth factors, mulcins have 00 been found that have an improved activity. For instance in the European patent application EP-131816 the described goal is to obtain a beta-Interferon with an improved biological activity and/or a less side-reactivity. Also various examples of chemical oC modifications are available: for instance the European application EP 236987 describes 00 the modification of L-2 to obtain a less toxic and less immunogenic substance with Simproved kinetics in clearing from the body. Patent application EP-0442724 describes PEG-ylated IL-6 which is a product with a longer half-life and an enhanced biological Sactivity, Patent application WO 88/01511 describes succinylation of IL-2 whereby an Nc enhanced solubility is achieved. In non of these patent applications there is any modification strategy besides a trial and error approach in which random mild modification is used to obtain one or a few modifications on the molecule. In addition, substantial losses in purification can occur, which was also the case in the example of PEG-yladon of IL-6 where only a 10 yield of the desired protein was obtained. The localization of the modification was done in none of these inventions.
In present invention there is almost no loss and the localization of the modification can well be performed. In addition, gradual chemical modification makes it even possible to specifically modify 1 residue at I place in the molecule, as demonstrated in the examples I and 2.Although most modifications are performed with irreversible agents, also reversible agents can be used. This also enables the very specific modification of other residues.
Antagonists: In the European patent application EP-0413383AI an antagonistic effect of a human IL-3 mutant is mentioned. However in this case it concerns the relation of remaining biological activity as compared to its receptor binding capacity. Thus, a real suppression the activity of the native IL-3 was not shown.
In patent applications PCT/US93/ 1197 and PCT/US93/11198 al kinds of IL-3 mutants are claimed, but also in this case there is no support what so ever of a real inhibitory activity. In addition, it is not probable that the mutants with the lowest activity are also the best inhibitors, since the chance on structural distortion is much higher than the chance on specific elimination of the catalytic activity.
There are other real an agonists for receptors of bovine growth hormone (Endocrinology 130: COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 113:23 FAX COLLISON CO IP AUST CANBERRA 1016/095 7 0 S2284 (1992)), mouse interleukin-2(EMBO J. 11: 3905 (1992)) htu-in hepatocyte growth factor (Biochemistry 31: 9555(1995)), IL-l(Scand. J. Immuro!. 36: 27(1992)) and IL-4 Exp. Med. 178: 2213 (1993). In all these publications no modification Sstrategy was described; the antagonists were all by-products of siructure-function S 5 analysis research. In addition, the concentrations of inhibitor that were needed to obtain significant inhibition were on average a hundred fold higher than the native growth factor concentration. These are concentrations that are harmful to clinical value of these 00 antagonists.
oO 0 The only strategy to generate a growth factor antagonist has been used for human growth hormone (Science 256: 1677(1992)). This was based on the disruption of one of the two receptor binding sites of the hormone. Also in this case there was a decrease of a factor 50 in receptor binding capacity, therefore requiring a problematic excess of c inhibitor as compared to growth factor.
However, with present invention it is possible to obtain a clinically applicable inhibition, possibly even with an enhanced receptor binding capacity. This can be explained with the hypothesis that the growth factor itself performs some kind of catalytic activity after receptor binding. This hypothesis was supported by the fact that IL-3 contains a catalytic zinc-ion (Biochem. Biophys. Res. Commun. 187: 859 (1992)). It is not necessary that the growth factor contains complete catalytic center. It is very well possible that the catalytic center is completed only in the growth factor -receptor binding. Therefore the concerning chemical modification is directed as specific as possible towards residues that are directly or indirectly involved in such Zinc binding and/or catalysis and this without distortion of receptor binding ability. The starting material can be any protein or peptide containing substance. However, because the Zinc ions can protect against denaturation, it might be necessary to reversibly denaturate the molecule and to add chelating agents to remove this Zinc from the molecule. As an illustration, but not as an limitation of the invention the modification of IL-3 with lodo-acetate is described. At the concerning pH this modification is directed to alkylation of His-residues. The method however, can be performed easily also with other reagents by a person that is skilled in the an. The same can be stated about the modification of other amino acids that are involved in the catalytic activity and/or Zinc binding. Also these residues can be easily modified by other chemical ways or by molecular biological ways, for instance by using deletion or substitution mutations. In addition this is not limited to IL-3 alone: There is a significant homology among the various receptors of the cytokine-superfamily: For instance Interleukins 2-7 Epo and GM-CSF. On top of that specific Zinc binding has also been found for IL-2 IL-6 GM-CSF and gamma IFN. It is also conceivable that the invention can be applied to much more signal peptides and/or proteins, since for instance Insulin, COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:24 FAX COLLISON CO IP AUST CANBERRA I017/095 8 0 O human growth hormone and prolactin also have specific Zinc binding propenies. In addition, it has been found that for certain cell-lines IL-3 can be replaced by for instance o Insulin. Finally it is possible that alkylation of a growth factor also has an other OO mechanism in the generation of a antagonist or cell-inhibitor. Therefore, in general also N 5 alkylation, preferably with lodo-acetate, is to be regarded as a separate approach in the invention. Therefore all the above mentioned applications and/or modified substances and/or DNA-constructs and the use thereof are embodied within the scope of the 00 invention.
00 o STherapies aainst ADS o 10 As already discussed, the invention can possibly be applied to the fight against AIDS. It should provide a better way to combat AIDS than the current possibilities: In the patent USA 5215745 of Csatary et al, an aspecific method for immunotherapy against AIDS is described. In this case a means of an aspecific virus infection of avian paramyxovirus and/or avian rotavirus is used to enhance the number of CD4 positive cells. This can at best lead to a postponement of the disease because the newly formed CD4 positive cells will be infected by the HIV after a short time. In contrast, our invention leads to the effective cellular immune-response against retroviruses.
In patent number USA. 5081226 ofBerzofsky et al. a therapy is directed at a specific immune-response against retroviruses. In this case for instance antibodies against the HIV glysoprotein 160/120 complex are generated. In our research that corresponded to the invention this approach lead to the protection of the HIV and its integration in histiocytic cells, thereby even promoting the disease. As a result the method could not demonstrate efficacy.
For this reason the patent number USA 5256767 describes a virus subunit vaccine without envelope. The draw back of this method however, is the fact thai lipophylic nuclear pans can at best express in a very low concentration in the context of MHC and therefore do not provide sufficient protection.
Vaccines that are based on inactivated viruses combine the objections of the two aforementioned patents.
In contrast to the above described problems we describe in our example 7 that HTLV
B
(AIDS Research and Reference Reagent Program Catalog, NIH Publication No. 91- 1536, Bethesda, MD, USA) is eliminated in vivo at low levels of antibodies. From the oligoclbnal swarm HTLVIIlBa it is known that it causes a persisting retroviral infection in normal circumstances. This also was demonstrated by the HTLVIIIB infection of coworkers within the group of John Moore via a needle accident. (AIDS Res. Hum.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16'.24 FAX COLLISON CO IP AUST CANBERRA l~018/095 SRetrovir. 6: 307 (1990) and J. Clin. Invest. 91: 1987 (1992)). In contrast, from our c example it can be deduced that these viruses can be eliminated if the antibody levels are low. The generation of B-cells and the subsequent antibody production by these B-cells Sis stimulated by various growth factors (for instance IL-1-7 and IL-1 Therefore it is 00 5 logical that the therapy against AIDS can also be accomplished by using antagonists of these growth factors, Thus, also the treatment of AIDS is considered to be one of the applications of the invention.
00 00 o SExamples: C' Example 1: Gradual chemical modification of IL-3 with Acetic Anhydride to obtain an IL-3 with an enhanced biological activity or a changed stability- Materials and Methods: Chemical Modification: Acetic Acid, dioxane, lysine hydrochloride and MES were from Sigma. the modifying agent was from Fluka.
Buffers: Acetate/NaOH was used for modification at p-H 5.0, MES/NaOH was used for modification at pH 5.5, 6.0 and 6.5. At pH 7.0 an NaH 2
PO
4 NaOH buffer was used.
Ten times concentrated stock solutions were prepared and directly filtered trough a 0.22 micron filter.
The reaction mixture contained 50 mM buffer, 2 mg/m] hIL-3 and 3 mM Acetic anhydride or Succinic anhydride respectively. The 10 times concentrated stock solutions were prepared fresh at the day of the experiment. Modification of hIL-3 was performed overnight at 30 oC. After modification it was determined by SDS-electrophoresis that the IL-3 was not degraded.
Tests of biological activity MO-7 cells were a kind gift of dr. I.P. Touw (Erasmus University of Rotterdam, The Netherlands). RPMI culture medium was from Gibco (Paisly, supplemented Bovine Calf Serum was from Hyclone (Logan, Utah, USA.) The cell culture medium consisted of RPMI with 10 of the calf serum. During normal tissue culture of the cells also 100 ng/ml IL-3 was added.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:24 FAX COLLISON CO IP AUST CANBERRA 1I019/095 Firstly 10 fold stock solutions were prepared .consisting of serial 3 fold dilution'.
ranging from 10 microgram/ml to I ng/ml in cell culture medium, after mixing Sthoroughly. 25 microliter of the stock solution was added to 225 microliter cell culture medium, followed by incubation at 37 oC. For the 6-day tissue culture 2 x 105 M07 cells 0 5 /ml were used as an end concentration and for the 10 day culture 4 x 10 3 M07 cellsml were used. After the tissue culture, overnight tritium thymidine incorporation was used to determine biological activities. From at least 2 independent growth-response curves there 00 was a determination of the average (and range of) the concentrations that resulted in maximal stimulation. The relative activity of the modified substances was determined as the ratio of these 50 concentrations of the modified and the native IL-3 respectivey
[IL-
3 ,mdinfied50% [L-3ntive]50% The standard activity for the native IL-3 on day 6 was 1.0 million units/mg of protein (n=10, sigma(n.1) 20 On day 10 it was 0.2 N million units/mg of protein (n=10, sigma(n.]) 30 Results; All results regarding to the more precise characterization in terms of average number of groups, the specificity and the places of the modification on the molecule are described later in this patent description. The results of the biological tests are shown in table 1: Table I: Relative biological activities of acetylated IL-3.
Acetylatien at Average relative activity (and activity-range) in tissue culture After 6 days After 10 days pH 5.0 1.5 1.4 (0.8-1.5) pH 6.0 0.9 0.9 (0.7-1.1) pH 6.5 0.5 0.2 (0.2-0.3) pH =7.0 0.9 0.6 (0.6-0,6) From the table it can be concluded that there is a significant difference in relative activity between day 6 and day 10 after modification at pH 6.5 and pH 7.0. Therefore, it can be assumed that this demonstrates the generation of a substance with significantly lowered stability. At pH 5 there might be an enhancement of biologic activity. Further aspects are discussed in examples 5 and 6.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16'.24 FAX COLLISON CO IP AUST CANBERRA I020/095 11 O Example 2: N Gradual chemical modification of IL-3 with Succinic anhydride for the generation of an improved IL-3 with an enhanced activity or stability.
00 Chemical Modification: Acetic Acid, dioxane, lysine hydrochloride and MES were from Sigma. the modifying agent was from Fluka.
OO Buffers: Acetate/NaOH was used for modification at pH 5.0, MES/NaOH was used for 0 0 modification it pH 5.5, 6.0 and 6.5. At pH 7.0 an NaH 2
PO
4 NaOH buffer was used.
Ten times concentrated stock solutions were prepared and directly filtered trough a 0.22 micron filter.
The reaction mixture contained 50 mM buffer. 2 mg/ml hlL-3 and 3 mM Acetic anhydride Sor Succinic anhydride respectively. The 10 times concentrated stock solutions were prepared fresh at the day of the experiment. Modification of hlL-3 was performed overnight at 30 OC. After modification it was determined by SDS-electrophoresis that the IL-3 was not degraded.
Results: All results regarding to the more precise characterization in terms of average number of groups, the specificity and the places of the modification on the molecule are described later in this patent description. The results of the biological tests as performed in example-i) are shown in table 2: Table 2: Relative biological activities of Succinylated IL-3.
Succiny)ation at Average relative activity (and activity-range) in tissue culture After 6 days After 10 days pH 5.0 1.7 1.6 (1.3-2.5) pH 6.0 1.4 1.3 (1.3-1.4) pH 6.5 0.4 0.4 (0.4-0.5) pH 7.0 0.3 0.3 (0.2-0.3) From table 2 it can be concluded that succinylation at pH 5 results in a significant enhancement of the activity and succinylaion at a pH 6 results in a lower activity.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:25 FAX COLLISON CO IP AUST CANBERRA I021/095 12
O
SExample 3: c A method to chemically modify biologically active peptides or proteins for the generation Qof a protein or peptide antagonist.
00 Materials and methods: Urea, EDTA, MES and NaOH were from Sigma. the Na-Iodo-acetate was from Fluka.
Buffers: MES/NaOH was used for modification at pH 6.0. Ten times concentrated stock 00 solutions were prepared in 8 M urea and filter-sterilized directly thereafter with a 0.22 0 o micron filter. The reaction mixture contained 50 mM buffer, 5.5 M urea and 50 mM SEDTA. From these reagents 10 times concentrated stock solutions in 8 M urea were 10 made freshly on the day of the experiment.
0
C
N 1. Moderate chemical modification of hlL-3: lodo-acetate was added in a concentration of 3, 10 and 30 mM. The IL-3 concentration was 2 mg/ml. The modification was performed during 24. 48 and 72 hours at 37 oC and subsequently it was studied by native electrophoresis. After 2 days and 30 mM Iodoacetate there was maximal modification without severe distortion of the bands from the modified material (an indication of severe denaturation of the molecules). In this case less than 2 of the starting material was left Because in this case there was an expectation of a minimal biological activity without severe denaturation of the protein, this sample was used in further experiments. Subsequently, SDS electrophoresis was used to demonstrate that no degradation of the molecule was found after modification.
2. Partial chemical modification: In order to optimize the inhibitory capacity of the modified hIL-3, also partial modification was performed. For this purpose 1 mg/ml IL-3 was modified for 18 hours at 37 OC with 10, 30 and 100 mM of lodo-acetate. After native electrophoresis and coomassie staining the sample of 100 mM gave maximal modification without excessive distortion of the bands in the gel. circa 5 of the starting material was still present.
Since only this sample showed inhibitory activity in the activity tests, this sample was used in further experiments.
3. The activity- and inhibition assays: The activity tests were performed as described in example 1. Growth response curves 2) for both controls and alkylated IL-3 were made by 10 fold serial dilution's ranging from 1000 to I ng/ml. Inhibitory activity of the alkylated IL-3 was tested by performing the same titration of control IL-3, but now in presence of 3 ng/ml alkylated IL-3.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:25 FAX COLLISON CO IP AUST CANBERRA [022/095 13 0 In order to determine the maximal receptor binding capacity partially modified IL-3 was Stitrated in serial 7 fold dilution in presence of 3 ng/ml of native IL-3. The titration range Swas from I15g/ml to 0.1 ng/ml and the titration was performed on only 4000 M07 (1 cells/m) to exclude any starvation phenomena's.
00
(NI
Results: 00
O
0 In figure I it is shown that the modified IL-3 is capable to inhibit the control-IL-3 by a factor 10-100. In addition 3 ng/ml of the modified IL-3 is able to suppress thymTdine 0 incorporation of 30-100 ng/ml control IL-3 for 80-90 Therefore, the modified IL-3 0 does not only have an Inhibitory activity, it also has an enhanced receptor binding capacity. This is confirmed in the titration of partially modified IL-3 (figure Only 0.
ng of partially modified IL-3 is sufficient for almost 50 inhibition of 3 ng/ ml native IL-3.
Example 4: Method for gradual enzymatic exoprotease treatment of IL-3 for the generation of a modified IL-3 with a changed stability and/or activity.
Materils and methods: Exoprotease treatments have been performed with Cathepsine-C and with Carboxypeptidase-Y from Boehringer. 1 mg/ml of IL-3 was incubated for 18 hours at 37 OC in presence of the protease. Cathepsine-C was added in serial two fold dilution's in a range of 1/2 to 1/128 mg/ml. The other reaction conditions were as described by the manufacturer.
Biological activities have been determined as described in example 1.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:25 FAX COLLISON 8 CO IP AUST CANBERRA la023/095 14 Results: The approach lead to the results in table 3. The modifications that did not result in a change in biological activity are not shown: Table 3 Changed activities after gradual exoprocease treatment: Protease treatment culture [Protease] Average relative activity and range) in tissue (mg/mi) After 6 days After 10 days Carboxypeptidase Y 1/40 1/20 1/10 0.6 0.08 <0.05 (0.5-0.7) (0.07-0.10) (<0.05) 1.0 0.2 <0.05 (0.7-1.5) (0.13-0.21) (<0.05) Cathepsine C 1 32 1/16 1/8 1/4 (2-6) (3-6) (2-4) (24) (0.7-1.5) (0.9-1.1) (0.9-1.7) (0.9-2.1) From this table it can be concluded that treatment with Carboxypeptidase Y at a concentration of 1/40 and at 1/20 results in a substance with a relative activity that is lower at day 6 than at day 10. Therefore, this indicates a substance with a higher stability as compared to native IL-3.
It can also be concluded form the table that Cathepsine C significantly enhances the activity at day 6 but not at day 10. Therefore, this is a also substance with a lowered stability.
Example Localization of modifications in a peptide or protein by the combination of protease treatment and mass spectrometry.
Materials and methods: Protease treatment: COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:25 FAX COLLISON CO IP AUST CANBERRA 1024/095 0 For localization of the modified residues the modified material was dialyzed against the C' appropriate buffer and subsequently fragmented by Endo Glu-C or Endo Lys-C as described by the manufacturer (Boehringer Mannheim, Germany). The incubation was 4 overnight at 37 oC, 2 mg/ml h)L-3 and a protein to protease ratio of 00 Laser Desorption Mass Spectrometry (LDMS): Pre treatment: 0 Solutions of 2,5 dihydroxybenzoic acid (DHB, Mr=154.12; 10 g/l) in milli Q water were 00 made freshly before each experiment, Both the (un)modified IL-3 solutions and their 0 digests were diluted to 0.1 mg/ml. From these diluted solutions 0.5 p1 was mixed with 0.5 p1 DHB-solution on the target. Subsequently, the target was dried to air at room O temperature in a slow stream of air.
Mass Spectrometry: Matrix Assisted Laser Desorption mass spectrometry was performed on a Finnegan MAT Vision 200 laser desorption mass spectrometer, equipped with a pulsed nitrogen laser (337 nm, pulse width 3 ns). The sample was exited to just above the ionization threshold (106-107 W/cm 2 The acceleration voltage was 6.5 kV. The ions were post accelerated to the conversion dynode on 10 kV for the electron amplification. Standard accuracy was about 0.05 but this can deteriorate to 0.1-0.2 due to experimental conditions.
Resulhs Because the signal of the LDMS was still sufficient in spite of the higher molecular weights, it became possible to localize all modifications. Two examples are shown: 1- Localization of modification with Succinic anhydride (pH 5.0) by means of Endo Lys-C treatment and LDMS: At the pH 5.0, Succinic anhydride modification and subsequent Endo Lys-C digestion a peak shifted from 1085 d to 1185 d, also the peak at 1108 d (1085 23 from Na shifted to 1208 d. Based on the protease specificity and the amino acid sequence this 1085 d peak can only correspond Ala' Lys 10 Since modification of Lysl 0 would disable the digestion on this amino acid, there would not be any Alal LysI 0 -fragment at all. Therefore, the modified amine residue is the amino terminus Ala 1 COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:26 FAX COLLISON CO IP AUST CANBERRA l025/095 2- Detection of modification of Lys 28 with Acetic anhydride by both Endo Glu-C treatment with subsequent LDMS and Endo Lys -C treatment with subsequent
LDMS:
At the pH 7 Acetic anhydride modification and subsequent Endo Glu-C treatment the peak at 1598 d shifted towards 1640 d, This shift corresponded exactly with the mass of I acetyl group. Also in this case the amino acid sequence enabled the localization to Ile 23 Asp 36 Since Lys 28 is the only amine-residue in this fragment, it can be deduced that this is the modified residue. This was confirmed by the Endo-Lys digestion where a fragment of 5815 d emerged. This fragment can only be explained if Lys 2 8 is the modified residue, thus disabling the digestion after that residue.
The other modifications have been analyzed in a similar way, resulting in table 4: Table 4: Localization of modifications on IL-3: Number of modified groups after modification with Modified at pH: Acetic anhydride Succinic anhydride Ala Ala Lys 28 Lys 66 Lys 00oo: Lys 116: Ala Lys 28 Lys 66 Lys 100; Lys 116; Ala LysO Lys 28 Lys 66 Lys '00: Lys 116: >90 >90 ±45 ±20 ±40% +40 -40 >90 ±70 ±50 ±65 ±80 >90 ±20 ±55 ±35 +65% 40 Ala Ala I Lys 28 Lys 66 Lys 100: Lys 116: Ala I Lys 28 Lys 66 Lys 100; Lys 116: +45 ±20 540 9+50% ±90 Ala I Lys2 Lys 66 Lys '00: Lys 116: -70 90 COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:26 FAX COLLISON CO IP AUST CANBERRA Ii026/095 17 o The table shows that both modifications have the same target residues on the protein. The c only exceptions are that Acetic anhyJride has a slightly higher degree of modification at pH 5.5 and at pH 7 Lys 0 was partly modified in contrast to the Lys 10 of the SSuccinic anhydride modified material.
0 5 Table 4 also shows that Lys1l6 is at least partly protected at pH 7. Since a phosphate buffer was used at that pH, the possibility arises that a phosphate group is binding at that place and thereby shields the Lys 1 1 6 for modification. To test this hypothesis hIL-3 was 00 modified at 50 mM buffering substance, consisting of MES and Phosphate. Acetic
O
1 anhydride 2 and 3 mM respectively) was used for the modification at pH 6.8 which is well within the buffering range of both buffers. In presence of 10 mM or more phosphate there was protection of I group: Lys 11 6. At a phosphate concentration below 1 mM this o protection was absent. Since 10 mM is the physiological phosphate concentration, it can Cl be assumed that present localization method enabled the demonstration and localization of a biologically significant phosphate binding. Therefore it is very conceivable that an antagonist or cell-growth inhibitor can be generated by distortion of this phosphate binding. Therefore, also this is to be considered within the scope of the invention. It can also be stated that the residues Lys 28 and Lys 6 6 also had slight protection by the phosphate, suggesting a close proximity in the 3-D structure. Thus, in this way it can even provide structural information.
Finally it can also be stated that the gradual chemical modification can be performed with such minimal degree that a specificity can be accomplished that is not limited to some types of-residues, not to only amine-residues, but even to I amine-residue on the complete molecule, namely Ala i Therefore this specificity is also included in the claims.
Example 6; Quantitative structure function analysis research using gradual chemical modification, protease treatment and laser desorption mass spectrometry.
Materials and methods: QSAR-strategy: This example was demonstrated with lysine modifications on hIL-3. The strategy consists of 5 steps of which the first step concerns the gradual chemical modification of the protein. Although the micro-environment of the various residues in the 3-D structure is not known, differences can be expected on the amino acid sequence alone. Even more differences can b expected in the 3-D structure.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:26 FAX COLLISON CO IP AUST CANBERRA a027/095 18 rI-
O
O We investigated acylation reactions on h[L-3 (step These reactions only take place on uncharged Lys residues, enabling the gradual modification by a stepwise increase in the SpH of the modification-reactions.
oo 0, The second step is the monitoring of the modification reaction. In order to study a sufficient number of possible conditions so that the optimal conditions can be achieved, a N mild and sensitive method is needed. This method is native electrophoresis 00 (Electrophoresis 15: 251 (1994)). However, also electrospray mass spectrometry can o form a suitable alternative. This is demonstrated in figure 3: Electrospray Mass o Spectrometry of Succinylated IL-3 at pH 5-7. Especially the combination of both enables S 10 the demonstration of complete specificity on amine-residues.
0 c The third step is the confirmation of the overall structural integrity. Circular Dichroism Spectroscopy can be used for this purpose(Electrophoresis 15: 251 (1994)). Although small differences are not visible by this method, substantial structural changes like denaturation are clearly detected.
The fourth step is the characterization and localization of the modified residues, for which the following techniques were used: native digestion with specific protease's, electrophoresis, electrospray mass spectrometry, and LDMS. The reaction specificity was determined by the combination of native electrophoresis and electrospray mass spectrometry. Localization was performed with endoprotease's and LDMS. as described in the previous example.
The fifth and last step is the testing of biological activity of the various modified forms of the protein. After this determination of activity the real involvement of the various localized residues can be deduced.
Results Chemical modification, structural confirmation and monitoring of the reaction: Chemical modification of hIL-3 and the monitoring was performed with Succinic anhydride or Acetic anhydride as previously described. Subsequently, it was found by Circular Dichroism that Succinic anhydride modifications at a pH larger than 7 resulted in an overall structural change (denaturation). Therefore, only the modifications at or below pH 7 were used for further investigation.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:27 FAX COLLISON CO IP AUST CANBERRA a028/095 19 0 SCharacterization and localization of the modifications: See previous example, 0 SActivity tests of the various modified forms of the protein and localization of biologically O0 00 important residues: Both the methods and the results of the tests for biological activity are described in N f examples I and 2. The combination of these results and the results of the localization of 00 00 the modified residues Tables 1 and 2 and previous example) enables statements on 0 involvement of several residues. Hereby, an important change is from unmodified to Cl modified at pH 5, which is accompanied by an enhancement in activity. Other important o 10 changes are from pH 6 to pH 6.5 (activity decrease by a factor 2) and from 6.5 to 7 O (activity increase by a factor 2): Since Succinic anhydride modification at pH 5 is accompanied by the modification of only I group, namely the amino terminus (Alal), it can be concluded that this group has some kind of limiting or regulating action. The increase in activity has also been found in structure-function research with deletion mutants.(J. Biol. Chem. 266, 21310 (1991); Proc. Natl. Acad. Sci, USA. 89: 11842(1992)), but it was never assigned to the first residue alone.
With the differences between pH 6 and pH 6.5 there is a more complex pattern: For acetic anhydride the decrease in activity of a factor 2-4 was accompanied with a modification of Lys 28 of 45 to 70 of the groups, for Lys 66 of 20 %l to 50 of the groups and for Lys'
I
O from 40% to 65% of the groups. Finally, there occurred modification on Lysll 6 of 40 to 80 of the groups, which is a decrease of unmodified groups of 60 to a factor 3 difference. Since this difference correlates exactly with the activity decrease, Lys 16 is the best candidate for the biological activity. This is confirmed by the factor 3 lowered modification at pH 7 (in comparison with pH that is accompanied with a factor 3 increase in activity. Therefore, Lys t 16 is important for biological activity.
This was all confirmed by modification with Succinic anhydride. In this case there was no decrease in modification for the at pH 7 modified material as compared to the at pH modified material, Accompanying this phenomenon there was no enhancement of activity either.
Thus it has been demonstrated that the residue Lys t 16 and the amino-terminus are of biological significance, while the amino terminus seems to have an inhibitory or regulating influence, Lys 1 1 6 seems to be imponant for the biological activity. In addition.
Lys1 16 is also protected by phosphate, suggesting a phosphate binding by that residue.
Since the residue is also important for the biological activity of the interleukin, this COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:27 FAX COLLISON CO IP AUST CANBERRA la029/095
O
Ssuggests that phosphate-binding is of importance for the mode of action of IL-3 and if this process is of importance for IL-3. it can also be of importance for other peptides and d< proteins.
00 Summarized, this method enables the localization of biologically important residues and Cl 5 the demonstrated phosphate binding has also enabled the establishment and localization of a possibly important physiological process. Therefore, the invention also embodies the \modification of a protein or peptide to introduce a new, preferably antagonistic activity by 00 00 means of the manipulation of the phosphate binding of the protein or peptide.
0 0 0 0 Example 7: Lowered levels of antibodies lead to effective in vivo cellular resistance.
Materials and Methods: The test system consisted of human chimeric 4 week old "X-linked immunodeficient" Mice. the chimerism was induced with conditioning by total body irradiation (TBI), and transplantation of 4 million human peripheral blood lymphocytes gram of recipient.
The TBI of the CBA/N mice was 9 Gy gamma. These mice also received a blood supporting treatment in the form of 0.5 million autologous bone-marrow cells intravenously Comparable irradiation, processing of the human blood and the transplantation is described in the Eur. J. Immunol. 22: 197 (1992). The mice were injected intraperitoneally (ip) daily with 10.000 I.U. of human Interleukin-2 (Eurocetus, Amstelveen, Benelux). The infection was done ip 1 hour after the transplantation of the human cells with a dose that is 10 times the minimal dose, still infectious in the "infectious center test" or ICT.
The conditioned CBAIN mice were pretreated ip with 250 microgram of monoclonal antibody anti- HIV-1 GP13 (against the CD4-binding place) or the anti HTLVnB F58H3 directed at the V 3 -loop.
The ICT was performed in duplo with CB15 cells (Proc. Natl. Acad. Sci. USA. 89: 3116 (1992)). Ten thousand cells were plated per well and after 5-7 days an ELISA was performed on HIV p24 protein (Organon Technica, Oss, The Netherlands). The sacrifice of the animal was always within 2 weeks after transplantation thus eloping the production of antibodies (unpublished data). On the day of the sacrifice of the mice, the cells were rinsed from the peritoneal cavity with medium that contained heparin (Organon Technica. Oss, The Netherlands). On these cells the ICT was performed in presence of CB15 cells and 100 I.U. human Interleukin-2 rn of culture medium.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:27 FAX COLLISON CO IP AUST CANBERRA 1a030/095 21
O
o Titration was performed in duplo in a tiiration ravge from 2.5 million to 0 in serial 5 fold dilution's. After 5-7 days the ELISA-test was performed on the HIV-p24 protein in the supematant of the culture medium. As a control for the presence of CD4' cells FACScan Sanalysis was performed as described in the Eur. J. Immunol. 22: 197 (1992).
00 Results: The results are shown in Table u Table 5: Protection of HIVInB virus by antibodies: 00 00 o Antibody 1 IC x 104 at day 5 after transplantation at day 8 after transplantation 0 10 Average (Range) Average (Range)
O
none 2 >200 >200 GP13 4 (0.4-100) 20 (2-50) F58H3 >200 (>200) =>200 (0.5->200) The table shows that HTLV 1 B persists for the first 5 days under these circumstances.
However 8 days after the transplantation it appears to have been eliminated, even in abundant presence of human CD4+ceIls. This indicates that the transplanted human Tcells eliminate the virus. However if either of the specific anti-HIV-1 antibodies are administered to the chimeric mice, the virus did persist (Table demonstrating that the persistence of HIV-infection is caused by antibodies.
From this it can be deduced that the lowering of the antibody levels in HIV infected humans can enable the T-cells to eliminate the virus, thereby providing the cure of the infection. These antibody-levels can be lowered by suppression of B-cells. Therefore this B-cell suppression is an interesting field of applications for the growth factor antagonists.
There are also other possibilities for the suppression of the HIV infection which can also be used separately: 1. Plasmaphoresis, that results in the lowering of the antibody-level. The usual clinical practice is the complete substitution of the plasma. An experimental therapy for instance for myasthenia gravis is the so called selective recovery of the plasma. In this case the plasma of the patient is purified from the harmful antibodies before returning to the patient. This in vitro selection can also be used for HIV-reactive, by preference HIV-envelope reactive antibodies.
2. Leukophoresis, for the lowering of the number of B-cells. It is preferred to remove the B- cells that are HIV-reactive. The leukocytes can be removed totally from the HIV-infected person. This form of leukophoresis is a routine clinical COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:28 FAX COLLISON 8 CO IP AUST CANBERRA I031/095 22 0 0 practice for other diseases. For HIV infected persons however, it has never been described. Alternatively the selective return of the white blood cells without Bcells can very easily be done. Selection can also consist of positive selection for T-cells or sib populations thereof.
0 5 3. In viv o depletion of antibodies. The invention also includes in vivo depletion by formation of immune complexes, as well selective as non-selective. Non selective removal is preferably done by antibody-specific antibodies. Selective removal is 00 preferably done by virus, inactivated virus, virus-subunits and/or virus-like or o identical proteins or peptides. These substances are preferably coupled to o 10 substances that promote clearing from the body.
4. In vive depletion of B-cells. This in vivo depletion can be performed trough o non-selective removal with B-cell specific antibodies, by preference with B-cell C' i apoptose-inducing antibodies. This can also be performed with bi-specific antibodies, preferably of the combination CD 9/CD3-reactivity. This has already been used in a phase I clinical trial in the Academic Hospital Utrecht (Utrecht, The Netherlands) for patients with B-cell tumors. This therapy results in a very substantial depletion of the number of B-cells (Personal communication: F.A. van Houten Academic Hospital Utrecht, The Netherlands). Selective removal of Bcells is preferably done by virus, inactivated virus, virus-subunits and/or viruslike or identical proteins or peptides or by antibodies. Preferably, these substances are coupled to B-cell depletion promoting substances.
Other methods that suppress the in vivo production of antibodies. An example is the use of transforming growth factor beta (TGF-beta).
HIV and other viruses integrate as provirus in the host genome. Therefore it can be present in these cells in al latent state for a long time. Therefore, in this invention it is preferred to activate such a provirus, preferably by administration of IL-2 to the host.
HrV persists in the histiocytic cells and these can produce low concentrations of virus that might escape recognition by the hosts immune-system. Therefore it is preferred to prolong the treatment for at least the life-time of these cells, In addition it is preferable to simultaneously perform passive immune-therapy, preferably with immunoglobulins of subjects that are not HIV-infecled. Therefore, this passive immune therapy is to be considered within the scope of the invention.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:28 FAX COLLISON CO IP AUST CANBERRA a032/095 Although this invention is described in a manner that is based on the limited knowledge of retroviruses like HIV. it is clear that several modifications can be made without diverting from the scope of the invention.
COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28
Claims (5)
- 3. A modified signal substance according to any one of claims 1-2, wherein at least one receptor binding place has an increased function, preferably an increase by at least a factor 2 or an increase by at least a factor 3 or an increase by at least a factor 10 or an increase by at least a factor
- 4. A modified signal substance according to any one of claims 1-3 with super-agonistic activity wherein there is an unchanged secondary amino acid backbone structure, and wherein the modification is a chemical modification and wherein the modification is a minimal chemical modification, and wherein the modification is at one or more solvent accessible residues and wherein the modification is at a specified position or at specified positions.
- 5. A modified signal substance according to any one of claims 1-4 wherein the modification is on a solvent accessible amine residue.
- 6. A modified signal substance with super-agonistic activity according to any one of claims 1-5 wherein the biological activity ranges from 40 of the natural counterpart, to 250 of the natural counterpart.
- 7. Use of a modified signal substance according to any one of claims I to 6 or a nucleic acid sequence encoding it for the preparation of a COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28 28/02 2007 16:28 FAX COLLISON CO IP AUST CANBERRA 1034/095 medicament for application in a treatment comprising stimulating stem cell replication or gene therapy. COMS ID No: SBMI-06429525 Received by IP Australia: Time 17:14 Date 2007-02-28
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| AU2007200889A AU2007200889A1 (en) | 1994-08-31 | 2007-02-28 | Gradual modification super-agonists and antagonists of signal-proteins and peptides |
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