MEMBRANE DISRUPTIVE PEPTIDES COVALENTLY OLIGOMERIZED

Abstract: The present invention relates to a modified membrane disruptive peptide. The present invention also relates to delivery complex comprising the modified membrane disruptive peptide and the use of the delivery complex. The modified membrane disruptive peptide is modified to form a covalently linked multimer.

Description: MEMBRANE DISRUPTIVE PEPΗDES COVALENTLY OLIGOMERIZED The present invention relates to a modified membrane disruptive peptide. The present invention also relates to a delivery complex comprising the modified membrane disruptive peptide and the use of the delivery complex. There is a demand in the field of gene therapy for simple non- viral gene delivery agents that can be used to efficiently transfect cells. Cationic peptides condense DNA by electrostatic interaction and have been used to prepare simple and reproducible peptide:DNA formulations. Methods for their preparation and purification are well documented. Cationic peptides have also been used as chemical anchors for the introduction of further peptidic or non-peptidic entities to assist in the passage of a packaged gene from an extracellular location to the nucleus of a desired target cell. Several groups have demonstrated that, when using cationic peptides as delivery agents in vitro, transfection is optimal only in the presence of exogenous agents that are believed to act by increasing the ability of genes undergoing transportation to escape from intracellular vesicles, which encapsulate them during or after cell uptake. Examples of such exogenous agents are chloroquine or membrane disruptive peptides. It is believed that destabilisation of biological membranes is central to the role of these agents. A breakdown in the structure of any membrane acting as a barrier to the passage of a delivery complex would increase its ability to access the nucleus. However, it is reasonable to assume that for many in vivo applications the use of exogenous agents such as the small organic molecule chloroquine, apart from complicating the formulation, would be hindered by the different in vivo clearance mechanisms and diffusion rates found between small molecules and macromolecules. Fusogenic or membrane disruptive peptides such as those described by Wagner (Wagner et al, PNAS, 89, 7934-8, 1992) or Smith (International Patent Application WO 97/35070) can be anchored either to condensing structures prior to complex formation or to pre-formed peptide:DNA complexes but this would also complicate manufacture of the formulation. Moreover, such peptides have been shown to be less efficient than adenovirus particles at enhancing gene transfer (Gottschalk et al., Gene Therapy, 3, 448-457, 1996; Wagner et al, PNAS, 89, 7934-8, 1992) and so the search continues for more effective peptides. A peptide that can both condense DNA and disrupt biological membranes would simplify the formulation and can lead to more efficient transfection. Melittin is a well known membrane disruptive peptide; however, despite being one of the most extensively studied peptide sequences, there appear to be only two instances in which its use in gene delivery has been described. Firstly, in the form of dioleoyl phosphatidylethanolamine-N-[3-(2-pyridyldithio) propionate] (a DOPE derivative) linked to melittin (Legendre et al, Bioconjugate Chem., 8, 57-63, 1997), and secondly in US patent US-A-5,547,932. In Legendre et al, the conjugation of a lipid is an expensive and complicating step and problems have been reported with the use of DOPE in vivo. In US patent US-A-5,547,932 formulations of an endosomolytic agent attached to a nucleic acid binding agent are described. The formulations described form relatively inefficient transfection formulations which are possibly also toxic to the cell being transfected. Indeed, the toxicity of melittin may explain why it has not been used as a lone peptide in a successful non- viral formulation. The present invention provides a modified membrane disruptive peptide, wherein the membrane disruptive peptide has been modified to form a covalently linked multimer. Preferably, the modified membrane disruptive peptide is further modified to form a substantially continuous α helix. By modifying the membrane disruptive peptide so that it forms a multimer and preferably so that it forms a substantially continuous α helix, it has been found that the toxicity of the peptide is reduced and that the peptide gives increased levels of transfection when used to deliver nucleic acids to cells. Without being bound by any one theory, it is proposed that the membrane disruptive peptide must be in the form of a monomer in order to be able to insert into a membrane where it may aggregate to form a pore. The formation of the pore is believed to be toxic to the cell as it enhances passive ion permeability. The multimerisation of the peptide prevents the formation of the monomer and thereby prevents pore formation and reduces cell toxicity. The multimers still retain some membrane disruptive properties due to predominantly non-polar amphiphilic α helices. By modifying the peptide so that a substantially continuous α helix is obtained, the membrane disruptive properties of the peptide are increased as the substantially continuous α helix is available to interact with cell membrane. The multimerisation of the peptide also has the advantage that the peptide is less likely to become dissociated from any bound nucleic acid. The term "a membrane disruptive peptide" means a peptide that is capable of promoting membrane destabilisation and lowering the energy required for a molecule to traverse the membrane. Assays such as the erythrocyte lysis assay can be used to determine if a peptide is a membrane disruption peptide; however, different membrane disruption peptides have different cell specificities and may lyse a different cell type to an erythrocyte. Accordingly, other cell types should be used in lysis assays to determine if a peptide is a membrane disruption peptide. The membrane disruptive peptide is preferably a toxic membrane disruptive peptide. It is further preferred that the membrane disruptive peptide is toxic by inserting itself into a membrane in the form of a monomer. It is further preferred that the membrane disruptive peptide is a toxic α helical membrane disruptive peptide such as melittin, cecropin A, cecropin PI, cecropin D, magainin 2, bombolittins or pardaxin (Saberwal et al, Biochimica et Biophysica Acta, 1197. 109-131, 1994). The membrane disruptive peptide preferably forms an amphiphilic α helix with one face rich in cationic residues, which enable condensation of DNA, and a hydrophobic face that is able to interact with membrane lipids. It is also preferred that the membrane disruptive peptide comprises a α helix region and a basic region at the C-terminus of the peptide, which can condense a nucleic acid. Preferably the membrane disruptive peptide is not the human bactericidal/permeability-increasing protein (BPI) (Gray et al, J. Biol. Chem., 264. 9505, 1989 and US patent US-A-5,856,302). Preferably, the membrane disruptive peptide is melittin. The term "peptide" as used herein refers to a polymer of amino acids having a chain length of between 10 and 150 amino acids. The term does not refer to or exclude modifications of the peptide, for example, glycosylations, acetylations and phosphorylations. Included in the definition are peptides containing one or more analogs of an amino acid (including for example, unnatural amino acids), peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and synthesised. The term "modified to form a multimer" means the membrane disruptive peptide is modified so that it is covalently linked to one or more other membrane disruptive peptides. Preferably the multimer is a dimer, trimer or a tetramer, more preferably a dimer or a tetramer and most preferably a dimer. The membrane disruptive peptides forming the multimer may be the same or different. The membrane disruption peptides can be linked together at any point along the length of the peptides; however, it is preferred that the peptides are linked together at the N-terminus end and/or C-terminus end of each peptide and most preferred that the peptides are linked together at the N-terminus end of each peptide. Preferably, the membrane disruptive peptide is modified to form a dimer by replacing an amino acid of the membrane disruptive peptide with an amino acid that forms a covalent bond directly via a disuphide bond or via linking group, with an amino acid of another modified membrane disruptive peptide. Alternatively, an amino acid that forms a covalent bond directly via a disulphide bond or via a linking group can be added to the membrane disruptive peptide. Preferably the amino acid added to or replacing an amino acid of the peptide is a cysteine amino acid, which can form a disulphide bond with a cysteine residue of another peptide. The membrane disruption peptides forming the dimer do not have to be identical; however, it is preferred that the peptides forming the dimer are identical. Preferably, the membrane disruptive peptide is modified to form a trimer or a tetramer by replacing two or more amino acids with two or more amino acids that form a covalent bond directly via a disulphide bond or via a linking group with an amino acid of other modified membrane disruptive peptides to form a trimer or a tetramer. Alternatively, two or more amino acids that form disulphide bonds directly or via a linking group can be added to the membrane disruptive peptide. The membrane disruption peptides forming the trimer or tetramer do not have to be identical; however, it is preferred that the peptides forming the trimer or tetramer are identical. The membrane disrupting peptides may be linked together via a linker such as commercially available linkers including bismaleimide or bisvinylsulphone linkers. The term "a substantially continuous α helix" means the region of the membrane disruptive peptide that forms a α helix and does not comprise one or more amino acids that disrupt α helix formation. Preferably the substantially continuous helix is at least 10 amino acids in length. Preferably the substantially continuous helix forms at least 5% of the length of the modified membrane disruption peptide, more preferably between 40% and 90% of the length of the modified membrane disruption peptide and most preferably about 80% of the length of the modified membrane disruption peptide. The presence of a α helix in a peptide can easily be measured using standard circular dichroism analysis. Amino acids that can disrupt α helix formation include proline, glycine, tyrosine, threonine and serine. However, as will be appreciated by one skilled in the art, the ability of an amino acid to disrupt α helix formation is dependent on the overall sequence of the peptide and other factors such as the pH of the solution in which the peptide is folded. Preferably the amino acid that can disrupt α helix formation is proline. Preferably, the membrane disruptive peptide is modified to form a substantially continuous α helix by replacing an amino acid, which disrupts α helix formation with an amino acid that does not disrupt α helix formation. In a preferred embodiment, the modified membrane disruptive peptide of the present invention has the amino acid sequence CIGANLKNLTTGLAALISWIKRKRQQ. It is further preferred that the modified membrane disruptive peptide forms a dimer via a direct disulphide linkage between the Ν-terminal cysteine amino acids. The modified membrane disruption peptide of the present invention can be further modified by the addition of other functional groups such as lipids, targeting groups such as antibodies or antibody fragments which target the peptide to specific cell types, antigenic peptides, sugars and neutral hydrophilic polymers such as PEG and PNP. Suitable functional groups, which can be added to the modified membrane disruption peptide of the present invention, are described in International Patent Application WO 96/41606 as well as methods for attaching such groups to a peptide. Preferably, the modified membrane disruption peptide of the present invention is further modified by the addition of amino acids to the substantially continuous α helix region of the peptide, wherein the additional amino acids extend the length of the substantially continuous α helix region. Preferably, the substantially continuous α helix region is extended so that the α helix region is at least 10 and more preferably at least 20 amino acids in length. Preferably, the modified membrane disruption peptide of the present invention is further modified by the addition of basic amino acids to the C-terminus region of the peptide. The C-terminus region of the modified membrane disruption peptide of the present invention is the region extending from the C-terminal amino acid to the region forming the substantially continuous α helix region. Basic amino acids are well known to those skilled in the art and include lysine, arginine and histidine. Preferably, the C-terminus region is modified by the addition of between 1 and 50 basic amino acids, more preferably between 5 and 15 basic amino acids. The present invention also provides a functional homolog of the modified membrane disruption peptide of the present invention. Preferred functional homologs of the modified membrane disruption peptide of the present invention, are those that still retain their activity and preferably have a homology of at least 80%, more preferably 90% and most preferably 95% to the peptide of the present invention. Preferably such functional homologs, which include fragments of the peptide of the present invention, differ by only 1 to 10 amino acids. It is further preferred that the amino acid changes are conservative. Conservative changes are those that replace one amino acid with one from the family of amino acids which are related in their side chains. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the peptide. However, it is sometimes desirable to alter amino acids in order to alter the biological activity of the peptide. For example, mutations which abolish or enhance one or more of the functions of the peptide can be particularly useful. Such mutations can generally be made by altering any conserved sequences of the peptide. It is preferred that such homologs have a homology of at least 80%, more preferably 90% and most preferably 95% to the protein or a fragment thereof of the present invention. Preferably such altered proteins or fragments thereof differ by only 1 to 10 amino acids. The present invention also provides the use of the modified membrane disruption peptide of the present invention in a delivery complex to deliver a nucleic acid to a cell. The modified membrane disruption peptide of the present invention can be used to deliver a negatively charged polymer, preferably a nucleic acid, to any cell type. Preferred cell types include prokaryotic cell types such as E. coli and eukaryotic cell types such as mammalian cells, including ex vivo primary cells, such as, HUNΕC and DC cells mammalian cell lines including HeLa, HepG2, CHO and myeloma cell lines, and lower eukaryotic cell types such as yeasts. Preferably, the modified membrane disruption peptide of the present invention is used to deliver a nucleic acid to a mammalian cell. The present mvention also provides a delivery complex comprising the modified membrane disruption peptide of the present invention and a nucleic acid. Preferably, the delivery complex consists of a nucleic acid to be delivered and the modified membrane disruption peptide of the present invention. The delivery complex of the present invention may be any delivery complex which comprises a nucleic acid to be delivered and the modified membrane disruption peptide of the present invention. Numerous dehvery complexes for delivering a nucleic acid to a cell are well known to those skilled in the art. Peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when co-administered with polylysine DNA complexes (Plant et al, 1994, J. Biol. Chem.. 269: 12918-24); Trubetskoy et al, 1992. Bioconiugate Chem.. 3: 323-327; Mack et al, 1994, Am. J. Med. Sci.. 307: 138-143) suggest that cocondensation of polylysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency. WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer. In a preferred embodiment, the dehvery complex of the present invention comprises the modified membrane disruption peptide and the nucleic acid encapsulated within nano- or a micro-particles such as polylactide glycolide particles and liposomes etc. In a f rther preferred embodiment the delivery complex of the present invention comprises a nucleic acid, a nucleic acid condensing peptide and the modified membrane disruption peptide of the present invention. The nucleic acid condensing peptide can be any peptide that condenses nucleic acids including polylysine and histone derived peptides. Preferred nucleic acid condensing peptides are described in International Patent Applications WO 96/41606 and WO 98/35984. Preferably the delivery complex is formed by condensing the nucleic acid with the nucleic acid condensing peptide to form a condensed nucleic acid complex. The condensed nucleic acid complex is then coated with the modified membrane disruptive peptide of the present invention. The present invention provides a method for forming a delivery complex according to the present invention comprising: 1. condensing a nucleic acid with a nucleic acid condensing peptide to form a condensed nucleic acid complex; and 2. coating the condensed nucleic acid complex with a modified membrane disruptive peptide according to the present invention. It has been found that the presence of serum during transfection increases the level of transfection. Preferably the serum is foetal calf serum. Other suitable serums that could be used are well known to those skilled in the art and include normal human serum and normal mouse serum. The modified membrane disruptive peptide of the present invention can be used to deliver therapeutic nucleic acids to cells in vivo, in vitro and for ex vivo treatments. The therapeutic uses of nucleic acids in a variety of diseases is well known to those skilled in the art. The therapeutic nucleic acid to be delivered to cells can be any form of DNA or RNA vector, including plasmids, linear nucleic acid molecules, ribozymes and deoxyribozymes and episomal vectors. Expression of heterologous genes has been observed after injection of plasmid DNA into muscle (Wolff J. A. et al, 1990, Science.247: 1465-1468; Carson D.A. et al, US Patent No. 5,580,859), thyroid (Sykes et al, 1994, Human Gene Ther.. 5: 837-844), melanoma (Nile et al, 1993, Cancer Res.. 53: 962-967), skin (Hengge et al, 1995, Nature Genet.. 10: 161-166), liver (Hickman et al, 1994, Human Gene Therapy. 5: 1477-1483) and after exposure of airway epithelium (Meyer et al, 1995, Gene Therapy.2: 450-460). Useful therapeutic nucleic acid sequences include those encoding receptors, enzymes, ligands, regulatory factors, and structural proteins. Therapeutic nucleic acid sequences also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Therapeutic nucleic acid sequences useful according to the invention also include sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids). Proteins or polypeptides encoded by the nucleic acid include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens and bacterial antigens. Specific examples of these compounds include proinsulin, growth hormone, dystrophin, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factor TGF-α, TGF-β, PDGF, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), matrix proteins (Type IN collagen, Type Nπ collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, Rb gene product, cytokines (e.g. 11-1, EL-6, IL- 8) or their receptors, viral capsid protein, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immunologic response, and other proteins of useful significance in the body. The compounds which can be incorporated are only limited by the availability of the nucleic acid sequence for the protein or polypeptide to be incorporated. One skilled in the art will readily recognize that as more proteins and polypeptides become identified they can be integrated into the delivery complex of choice, transfected using the modified membrane disruption peptide of the present invention and expressed. Nucleic acids delivered using the modified membrane disruption peptide of the present invention include those that encode proteins for which a patient might be deficient or that might be clinically effective in higher-than-normal concentration as well as those that are designed to eliminate the translation of unwanted proteins. Nucleic acids of use for the elimination of deleterious proteins are antisense RNA and ribozymes, as well as DNA expression constructs that encode them. Ribozymes of the hammerhead class are the smallest known ribozymes, and lend themselves both to in vitro synthesis and delivery to cells (summarized by Sulhvan, 1994, J. Invest. Dermatol.. 103: 85S-98S: Us an et a . 996. Curr. Onin. Struct. Biol.. 6: 527-533). The present invention also provides the modified membrane disruptive peptide or the delivery complex of the present invention for use in therapy. The present invention further provides the use of the modified membrane disruptive peptide or delivery complex of the present invention in the manufacture of a composition for the treatment of a genetic disorder. A genetic disorder is defined as a disorder that can be treated at the genetic level i.e. by delivering a nucleic acid to the patient in need of such treatment. Genetic disorders include, but are not limited to, enzymatic deficiencies (e.g. those of the liver, digestive system, skin and nervous system), endocrine deficiencies (e.g. deficiencies of growth hormone, reproductive hormones, vasoactive and hydrostatic hormones), exocrine deficiencies (such as deficiencies of pancreatic hormone secretion), neurodegenerative disorders (such as Alzheimer's Disease, amyotrophic lateral sclerosis, Huntington's disease, Tay Sachs' disease, etc.), cancer, muscular dystrophy and albinism. The present invention also provides a method of treating a genetic disorder comprising administering to a patient in need of such treatment an effective dose of a delivery complex comprising a therapeutic nucleic acid and the modified membrane disruptive peptide of the present invention. The present invention is now illustrated in the appended examples with reference to the following figures. Figure 1 shows the erythrocyte lysis activity of CP36 dimer (CP36D), CP36 monomer (CP36M) and melittin (CP1). Figure 2 shows the erythrocyte lysis activity of CP36 and CP48. Figure 3 shows the transfection of HepG2 cells when transfected with CP1 2, 18, 36, 39, 41, 42, 43, 44, 45, and 48 complexed at various charge ratios in the presence or absence of DTT and in the presence of 10% foetal calf serum. Figure 4 shows the protein levels of HepG2 cells when transfected with CP1 2, 18, 36, 39, 41, 42, 43, 44, 45, and 48 complexed at various charge ratios in the presence or absence of DTT and in the presence of 10% foetal calf serum. Reduced protein levels are indicative of a toxic effect on the cells. Figure 5 shows the transfection of HepG2 cells with pCMNβ complexed to CP36/1 (first batch), CP36/1.2 (second batch) in the presence or absence of DTT and in the presence or absence of 10% foetal calf serum. The nomenclature Red means in reduced form i.e. in the presence of 5mM DTT; and ΝR means in non-reduced form i.e. without added DTT. Figure 6 shows data from an experiment comparing luciferase expression from HUNEC transfected with CP36 complexed-DΝA from different batches of CP36 peptide, at four charge ratios. Batches of peptide refer to different syntheses and were termed CP36/1, /3, 14, 15, 16. Figure 7 shows a compilation from a number of experiments (no. of experiments shown in brackets) comparing average luciferase expression from HUNEC transfected with CP36 complexed-DΝA and PEI complexed-DΝA at various charge or Ν:P ratios, PEI used was 22kDa linear PEI (Exgen-500). Figure 8 shows a compilation from a number of experiments (no. of experiments shown in brackets) comparing average % cells expressing GFP from HUNEC transfected with CP36 complexed-DΝA and PEI complexed-DΝA at various charge or Ν:P ratios. PEI used was 22kDa linear PEI (Exgen-500). Figure 9 shows luciferase expression in vivo after dosing with 75μg DNA using various peptides in a number of tissues. Figure 10 shows the average luciferase expression from HUNEC transfected with ΝBC28 :DNA particles coated with CP36 dimer. EXAMPLES Materials and Methods Peptide synthesis and purification Method 1: Multiple Peptide Synthesiser (MPS ~ ) The peptides were synthesised on a P.E. Biosystems Pioneer peptide synthesiser running version 1.7 of the instrument software. The instrument was equipped with a single MPS unit attached to column position 2, controlled by a workstation running software version 1.3. Each synthesis was carried out on a 0.05mmol scale using Fmoc-PAL-PEG-PS resin (P.E. Biosystems). For the synthesis the extended, slow activation and coupling cycles were used which were provided with the instrument. Deprotection was carried out using a solution of 20% Piperidine in DMF. The following amino acid derivatives were used as appropriate for the peptide; Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L- Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L- Trp(Boc)-OH, Fmoc-L-Nal-OH, Fmoc-L-Lys(Fmoc)-OH. Activation of the amino acids was achieved using TBTU and DIPEA. In the final synthesis step the Fmoc group was removed followed by solvent exchange into dichloromethane and drying of the resin under a stream of dry nitrogen. The columns containing the resin were then taken and dried further under vacuum for 2-3 hours at room temperature. The peptides with no cysteine residues were cleaved from the resin using trifluoroacetic acid (TFA)/Water/Triisopropylsilane(TIS) 95:2.5:2.5 and those containing cysteine residues were cleaved using trifluoroacetic acid (TFA)/ Water/ Triisopropylsilane (TIS)/ ethanedithiol (EDT) 92.5:2.5:2.5:2.5. An empty 5ml syringe was attached to one end of each column and a 2.5ml syringe containing the cleavage mixture was attached to the other end. Cleavage mixture (1ml) was injected onto each column and they were left to stand for 30 mins, then a further 1ml was injected and after 30 mins the last 0.5ml. After standing for 30 mins the empty 2.5ml syringe was removed and the remaining cleavage mixture drawn into the 5ml syringe. The column was removed from the 5ml syringe, inverted and replaced, then a fresh 2.5ml syringe containing cleavage mixture attached to the other end and the cleavage procedure repeated. The contents of the 5ml syringe were then expelled into a 50ml centrifuge tube containing diethylether (45ml). The resulting precipitate was allowed to settle for 1-2 hours at room temperature and the supernatant was poured off. The remaining diethylether was blown of under a stream of dry nitrogen gas and the pellet dried further under vacuum for 2-3h. Method 2: Continuous flow synthesis of the Peptides. The peptides were synthesised on a P.E. Biosystems Pioneer peptide synthesiser running version 1.7 of the instrument software which was controlled by a workstation running software version 1.3. Each synthesis was carried out on a 0.2mmol scale using Fmoc-PAL-PEG-PS resin (P.E. Biosystems). For the coupling of all the amino acids except for cysteine, extended coupling cycles were used which were provided with the instrument. For the coupling of cysteine a special extended (1 hour coupling time) solvent exchange cycle was used so that coupling took place in DMF/DCM 1:1. Deprotection was carried out using a solution of 20% Piperidine in DMF. The following amino acid derivatives were used as appropriate for the peptide: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L- Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Nal-OH, Fmoc-L-Lys(Fmoc)-OH. Activation of the amino acids except cysteine was achieved using TBTU and DIPEA in DMF. Activation of cysteine was achieved using TBTU in DMF and Sym-Collidine in DCM. In the final synthesis step the Fmoc group was removed followed by solvent exchange into Dichloromethane and drying of the resin under a stream of dry nitrogen. The resin was washed out of the column into a filter funnel allowed to air dry for a few minutes the transferred to a pre-weighed flask and dried further under vacuum until a constant weight was achieved. The peptides with no cysteine residues were cleaved from the resin using trifluoroacetic acid (TFA)/Water/Triisopropylsilane(TIS) 95:2.5:2.5 (20ml) for 1.5h at room temperature and those containing cysteine residues were cleaved using trifluoroacetic acid (TFA)/ Water/ Triisopropylsilane (TIS)/ ethanedithiol (EDT) 92.5:2.5:2.5:2.5 (20ml) for 1.5h at room temperature. In each case the resin was then filtered off, washed with neat TFA (3x5ml) and the combined filtrate and washes evaporated down to a volume of ca. 3ml. The residue was transferred to a 50ml centrifuge tube containing diethylether(45ml). The resulting precipitate was allowed to settle for 1-2 hours at room temperature and the supernatant was poured off. The remaining diethylether was blown of under a stream of dry nitrogen gas and the pellet dried further under vacuum for 2-3h. Purification of the peptides The peptides were purified by reverse phase h.p.l.c. The more polar peptides were purified on either a Shandon Hypersil SAS column (12θA, lOμ, 150x21.2mm) using a gradient of water(0.1 % TFA) Acetonitrile (0.1 % TFA) typically 10-65% Acetonitrile in water over 20 minutes with a flow rate of 24ml/min or a Phenomenex Jupiter C4 column (120 A, 5μ, 250x10 mm) using a gradient of water(0.1% TFA) Acetonitrile (0.1% TFA) typically 30- 100% Acetonitrile in water over 30 minutes with a flow rate of 6 ml min. The fractions containing the peptide of interest, as measured by matrix assisted lazer desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) and HPLC analysis were pooled and lyophilised. Peptide Multimer Formation For complete dimerisation or oligomerisation via a disulphide or cystine linkage, pure peptide was dissolved in fresh 20mM ammonium bicarbonate and left at room temperature for 16 hours. The dimerisation was confirmed by analytical gel filtration chromatography using a Pharmacia Superdex Peptide HR 10/30 column run in 20% acetonitrile in water containing 0.1% TFA, with a flow rate of between 0.8 and l.Oml/min. The decrease in elution time of the dimerised peptide was confirmed by monitoring absorbance of eluent at 214nm. For the synthesis of bisvinylsulphone linked conjugates (CP48), 2.0mg Biolink 6 (Molecular Biosciences, Colerodo) was dissolved in lOOμl acetonitrile and made up to a 1.0ml volume using 25mM HEPES, pH7.2. 200μl of this solution was then added to 15 mg of CP36 monomer (5.2 μmol) dissolved in 800μl buffer. The reaction was monitored by MALDI and RP-HPLC and judged complete after 1 hour at 24°C. The final product was isolated by purification of Phenomenex Jupiter C 4 semipreparative column using a 30 to 100% gradient of increasing acetonitrile in water containing 0.1% TFA over 30 minutes. The mass of the final product was confirmed by MALDI-TOF MS. Erythrocyte Lysis Assay To 9.0 ml blood, 1.0 ml 110 mM citrate, pH5, was added to stop coagulation. The blood was spun at 2000 rpm for 5 min. The plasma supernatant was aspirated and the pellet washed with HBS at least six times, by serial mixing, centrifugation and aspiration. The clear supernatant was aspirated and the pellet washed twice with the appropriate buffer: either HBS (pH7.4) or 15mM sodium acetate, pH5, 150mM NaCl. The appropriate buffer was added to the blood pellet to make a total volume of 6 ml and 1ml taken from this stock preparation and diluted 15 times with buffer to give the final working solution. The peptides were tested as serial dilutions in a 96-well plate in triplicate by adding 75ml of blood solution, in appropriate buffer, to 100ml of peptide solution in corresponding buffer, and mixing. The blood was incubated with the peptide for 1 h at 37°C. At this stage 1% Triton X-100 was added to blood solution containing no peptide to act as a control for 100% lysis. The cells were spun down at 2500rpm for 5 min and 80ml of supernatant taken for spectrophotometric analysis at 450nm. Preparation of Complexes Plasmid DNA at 40μg/ml in HBS (HEPES buffered saline) pH 7.4 was rapidly mixed with an equal volume of an appropriate concentration of transfection agent in HBS and allowed to incubate for up to 1 hour at room temperature. The concentration of transfection agent was determined for peptides by the final desired charge ratio, or for PEI (Exgen-500, Euromedex, France) by the final desired ratio of nitrogen (from PEI): phosphate (from DNA). The charge ratio was calculated according to the definition by Feigner et al (1997) (Nomenclature for synthetic gene delivery systems. Gene Therapy, (1997) 8:511-512). HepG2 Transfection Assay 1. β-Gal Transfection Protocol HepG2s were plated the day before transfection at 5 x 10 4 cells/well in a 96- well plate in DMEM + 10% FCS (with antibiotics) and incubated at 37°C. The next day the cells were washed with 100 μl/well PBS. 90μl HEPES buffered RPMI containing 10% FCS and antibiotics was added to the cells followed by lOμl transfection complex, prepared as described above comprising plasmid pCMNβ reporter plasmid (plasmid encoding for β- galactosidase). The complexes were formulated in the presence or absence of 5mM DTT to reduce all peptide disulphide bonds. Using a relevant control, it was confirmed that the presence of DTT in the formulation itself had no observable effect on transfection. Cells were transfected in triplicate with each complex. The cells were centrifuged at 1100 rpm, then incubated under humid conditions in a non-gassed incubator at 37°C for 5 h. After 5 h the transfection medium was removed and the cells washed with lOOμl/well PBS before incubation in DMEM/10% FCS (with antibiotics) medium for 19-20 h in a CO 2 -gassed incubator. Finally, the cells were washed twice with PBS, lysed with 30ml 0.1% Triton, 250mM Tris, pH8, and frozen at -20°C before β-gal assay. 2. β-Gal Assay The frozen lysed cells were thawed at room temperature and lOμl from each well removed for protein assay. The remaining cell lysate was assayed for β-gal reporter using a Galacton-Sstar β-gal assay (TROPIX) and luminescence was measured using a 96 well TopCount scintillation counter running in SPC mode. A DC protein assay kit (BioRad) was used to assay lysate for total protein content. The transfection counts were reported as pg β-gal/ ng total protein, using a β-gal standard curve prepared in cell lysate from untransfected cells on the same 96-well plate. HUVEC Transfection Assays Luciferase Transfection Assays Primary HUNECs (Promocell, Germany) were plated at lxl 0 4 cells/well the day before transfection in a 0.1% gelatin coated 96-well plate in Endothelial Growth Medium with Supplements (EGMS) (Promocell, Germany) and incubated at 37° in a CO 2 -gassed incubator. The next day cells were washed with PBS. 90μl per well medium M199 (Sigma) containing 10% FCS and antibiotics wastadded to the cells followed by lOμl per well transfection complex, prepared with pCMNlμic reporter plasmid (plasmid encoding for firefly luciferase). Cells were transfected in triplicate for each complex. The cells were centrifuged at 1100 rpm for 5 minutes, then 5 incubated for 1 hour at 37°C in a CO 2 - gassed incubator. After 1 hour the transfection medium was removed and the cells washed withlOOμl well PBS before incubation in 100μl/ ell EGMS for 20-24 hours at 37°C in a CO 2 -gassed incubator. T.nπiferase Assay After 20-24 hours Luciferase expression was determined using LucScreen assay (TROPIX) with luminescence measured using a 96-well TopCount scintillation counter (Packard) running in SPC mode GFP Transfection Protocol Primary HUNECs were seeded at 1.5xl0 5 cells/well the day before transfection in 2 ml of EGMS, in 6 well plates. The next day cells were washed twice with 2 ml per well PBS, and 1 ml per well of transfection solution was added, consisting of: lOOμl transfection complex prepared with pCMNEGFP reporter plasmid (plasmid encoding for green fluorescent protein) mixed with 900μl Ml 99 containing 10% FCS and antibiotics. Cells were transfected in duplicate for each complex. Cells were incubated for 2 hours at 37°C in a CO -gassed incubator, after which the medium was changed back to 2ml per well EGMS. The cells were incubated for a further 24 ours at 37°C in a CO 2 -gassed incubator. GFP Assay After 24 hours, cells were washed twice with 2 ml per well PBS, trypsinised and resuspended with Ml 99 media + 10%FCS. The % transfected cells was determined by FACS analysis. In Vivo Delivery of pCMVLuc Using Peptides pCMNLuc at 500μg/ml in HBS was mixed with an equal volume (typically 500μl) of peptide or polylysine (127mer) (Sigma) at the appropriate concentration to give the desired charge ratio. Charge ratio was as defined by Feigner et al (Hum. Gene Ther., %(5\ 511-2, 1997). Peptide or polylysine was added to DNA over 3-4 seconds whilst mixing on a vortex mixer at 800rpm. Complexes were incubated for 1 hour at room temperature. 300μl of complex was injected into the tail vein of CD-I mice. 20 hours later mice were sacrificed and 80-200mg of each organ was removed, briefly blotted to remove excess fluid, and frozen in liquid nitrogen and stored at -80°C. Frozen tissue was weighed and then thawed in lysis buffer (lOmM sodium phosphate, containing ImM EDTA, 1% Triton X-100, 15% glycerol, 8mM MgCl 2 , 0.5mM PMSF, ImM DTT), and homogenised for 0.3- 2 minutes using a Mini bead beater-8 (Stratech Ltd) and 1mm glass beads. The homogenate was removed and the glass beads washed with lysis buffer and the washings combined with the homogenate. Particulates were removed by centrifugation for 5 minutes at 13000rpm, and 80μl assayed for luciferase activity in a Berthold LB593 luminometer, using O.lmM luciferin, 0.44mM ATP, and a 4 second acquisition time. Results are expressed as RLU corrected to mg weight of each tissue. The following peptides were prepared as monomers, or where possible as dimers or other multimers using the methods described above: CP1 GIGANLKNLTTGLPALISWIKRKRQQ-CONH 2 CP2 CIGAVLKNLTTGLPALISWIKRKRQQ-CONH 2 CP18 GIGAVLKNLTTGLAALISWIKRKRQQ-CONH 2 CP36 CIGANLKVLTTGLAALISWIKRKRQQ-CONH 2 CP37 GIGANLENLTTGLAALISWLERERQQC-CONH 2 CP39 Nle-IGAVLKVLTTGLAALISWIKRKRQQ-CONH 2 CP41 CIGAVLKNLTTGLAALISWLKRKRQQ-CONHz CP42 CIGAVLKVLTTGLAALLSWLKRKRQQ-CONH 2 CP43 CIGAVLKNLTTGLWALISWLKRKRQQ-CONH 2 CP44 CIGAVLKNLTTGLAWLISWLKRKRQQ-CONH 2 CP45 CIGAVLKNLTWGLAALISWLKRKRQQ-CONH 2 CP48 CIGAVLKVLTTGLAALISWIKRKRQQ-NH 2 KVLTTGLAALISWIKRKRQQ-NH 2 CP-46 NH2-LLQSLLSLLQSLLSLLLQWLKRKRQQ-CONH2 CP-47 NH2-CLLQSLLSLLQSLLSLLLQWLKRKRQQ-CONH2 CP-49 NH2-CIGAVLKNLTTGLAALISWIKRKRQQC-CONH2 CP-50 NH2-GIGAVLKNLTTGLAALISWIKRKRQQC-CONH2 CP-51 NH2-CIGANLENLTTGLAALIS WLERERQQ-CONH2 CP-52 NH2-CIGAVLKVLTTGLAALISWIKRKRQQK-CONH2 I NH2-CIGANLKNLTTGLAALISWIKRKRQQ-CONH2 CP-53 NH2-GIGANLKNLTTGLAALISWIKRKRQQKC-CONH2 I NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2 CP-54 NH2-CIGANLKNLTTGLAALISWLAALISWIKRKRQQ-CONH2 CP-55 NH2-CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2 \ S s \ NH2-GIGAVLEVLTTGLAALISWLERERQQC-CONH2 CP56 NH2-CIGANLKNLTTGLAALISWIKRKRQQ-CONH2 S S NH2-CIGAVLKVLTTGLAALISWIKRKRQQK-CONH2 I NH2-CIGAVLKNLTTGLAALISWIKRKRQQ-CONH2 I s s I NH2-CIGANLKVLTTGLAALISWIKRKRQQ-CONH2 CP-57 NH2-GIGANLKVLTTGLPALISWIKRCRQQ-CONH2 CP-58 NH2-CIGANLKVLTTGL AALIS WIKRKRQQ-CONH2 \ S s \ NH2-GIGAVLKVLTTGLAALISWIKRKRQQC-CONH2 CP-59 NH2-GIGANLENLTTGLAALISWLERERQQC-CONH2 / S s / NH2-CIGAVLKVLTTGLAALISWIKRKRQQK-CONH2 I NH2-CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2 \ S s \ NH2-GIGAVLEVLTTGLAALISWLERERQQC-CONH2 CP-60 NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2 I NH2-GIGAVLKVLTTGLAALISWIKRKRQQKC-CONH2 S S NH2-GIGAVLKVLTTGLAALISWIKRKRQQKC-CONH2 I NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2 CP1 is melittin, the main toxic component of bee venom. CP2 is CP1(G1 to C). This peptide was designed to form N-terminal cysteine linked dimers ofCPl. CP18 is CP1(P14 to A). This peptide was modified to form a substantially continuous helix. CP36 is CP1(G1 to C; P14 to A). This peptide modified so that it forms a dimer and has a substantially continuous helix. CP39 is CP36(C1 to Norleucine). The modification was introduced to increase the hydrophobicity at the N-terminus. Peptides CP41 to CP45 were designed to study the effects of conserved amino acid changes on CP36 activity. CP37 is CP 18(120 to L) and all lysines are replaced with glutamates. CP41 is CP36(I20 to L). CP42 is CP36(H7 to L; 120 to L). CP43 is CP36(A14 to W; 120 to L). CP44 is CP36(A15 to W; 120 to L). CP45 is CP36(T11 to W; 120 to L). CP46 is a functional homolog of melittin containing conserved amino acid changes and is based on the sequence described by DeGrado et al, (1981), J. Am. Chem. Soc, 103. 679-681. CP47 is CP46 with an added N-terminal cysteine for dimer formation. CP49 is CP36 with an added C-terminus cysteine for multimer formation. CP50 is CP 18 with an added C-terminus cysteine for multimer formation. CP51 is CP41 with all the lysine residues substituted by glutamates. CP52 is a dimer of CP36 formed by bis-amine modification of an additional C-teιτninus lysine. CP53 is a dimer of CP36 with an added C-terminus lysine and a cysteine residue linked to CP36 CP54 is CP36 with LAALISW inserted at position 20 to increase the length of the α helix CP55 is a heterodimer of CP36 and CP51. CP56 is a tetramer consisting of CP52 and 2 CP36 peptides. CP57 is CP1(K23 to C). CP58 is a heterodimer of CP36 and CP50. CP59 is a tetramer consisting of a dimer of CP52 linked to two CP37 peptides. CP60 is a tetramer consisting of a dimer of CP53. CP61 is a dimer consisting of CP36 and CP51, wherein a disulphide bond is formed between the N- terminal cysteines. It was observed that although melittin (CPl) could effectively bind to DNA, as determined by plasmid retardation on an agarose gel, melittin:DNA complexes conveyed little or no transfection of HepG2 cells and that melittin was toxic to cells. After incubation of cells with such complexes, the total protein content per cell decreased with increasing ratios of melittin to DNA (figure 4). This implied a toxic effect caused by the ability of melittin to form numerous structures that are perturbing to biological membranes. Literature on the mechanism of melittin suggests that although the peptide exists as a tetramer in solution at high concentrations and/or high ionic strength, it must be able to dissociate into a monomeric form in order to be able to insert into a membrane, in which it can aggregate into a pore-forming tetramer. The pore forming tetramer of melittin is believed to enhance passive ion permeability and is considered to be toxic to cells. It was proposed that the dimerisation or tetramisation of melittin should result in a construct that would be less toxic to cells, the dimerisation or tertramisation effectively preventing initiation of pore- forming tetramer formation. Melittin dimers or tetramers would nevertheless possess membrane disruptive properties due to predominantly non-polar amphiphilic helices. It was decided to optimise the interaction between amphiliphilic helices of melittin and membrane surfaces by replacing Pro residues at position 14 of melittin, which causes a kink in helical structure, with a residue that would allow continuation of the helix. As indicated above, dimers were constructed and their membrane disruptive activity measured by an erythrocyte lysis assay. Surprisingly, although the lytic activity of dimers was greater than melittin at pH 7 and 5 (see Figures 1 and 2), the dimers appeared less toxic to mammalian cell lines (see Figure 4). It is therefore proposed that pore formation is toxic but other membrane disruption destabilisation mechanisms are not toxic. The dimerisation of melittin together with helix elongation, as described, resulted in constructs which could bind DNA; the resulting DNA complexes were significantly less harmful to HepG2 cells, as determined by protein content determination (see Figure 4), and conveyed efficient transfection of number of cell types in the absence of exogenous agents (see Figures 3 and 5). It was also found that the presence of foetal calf serum during transfection gave increased levels of transfection (see figure 5). Figure 9 shows that peptides CP36 and CP61 of the present invention are effective at increasing transfection of DNA to a number of tissues in vivo. Delivery Complex Coated with CP36 Dimer The delivery complex was prepared as follows. 1. Preparation NBC28:DNA complexes at charge ratio ±4 and DNA concentration of 25μg: Solutions of NBC28 at 92.6μg/ml and of pCMVluc DNA at 50μg/ml were prepared in lOmM HEPES pH7.4. The peptide solution was added to the DNA solution in a 1:1 (v/v) ratio and left to stand for 1 hour at RT. NBC28 is a nucleic acid condensing peptide having the amino acid sequence: TKXKKKKKKKKKKKKKKKKYCG. 2. Coating of the complexes: The coating peptide was added to the complexes followed by some lOmM HEPES buffer to make the volume up to 97% of the final volume required. The resulting complexes were vortexed for lOsec. then stored at 4°C overnight. 3. Salt spike: Next morning (ca. 18h. later) 5M salt (3% of the final volume to give 150mM salt) was added to the complexes^EcTiwere then vortexed for 10 sec, and left to stand for a further hour at RT. 4. Reconstitution of the complexes: The complexes, in 1.5ml Eppendorfs, were spun down for 30min. at 13000rpm in an MSE Microcentaur centrifuge. Supernatant (75%) was removed and an equivalent volume of fresh HBS was added with slow vortexing and sucking up and down with the Gilson pipette for 30 sec. 5. Sonication of the complexes The complexes, in 1.5ml Eppendorf tubes, were sonicated for 30sec. in a sonication bath prior to transfection. The complexes were then transfected into HUNEC cells in accordance with the method described above. Figure 10 shows that the complexes coated with CP36 dimer give good transfection of HUNEC cells. In particular, the level of transfection is greater than that obtained with CP36 or ΝBC28 alone.

Claims: CLAIMS 1. A modified membrane disruptive peptide, wherein the membrane disruptive peptide has been modified to form a covalently linked multimer. 2. The modified membrane disruptive peptide of claim 1, which is further modified to form a substantially continuous α helix. 3. The modified membrane disruptive peptide of claim 1 or claim 2 wherein the membrane disruptive peptide is a α helical membrane disruptive peptide and is toxic to cells. 4. The modified membrane disruptive peptide of any one of the preceding claims, wherein the membrane disruptive peptide is melittin. 5. The modified membrane disruptive peptide of any one of claims 2 to 4, wherein the membrane disruptive peptide is modified to form a substantially continuous α helix by replacing an amino acid which disrupts α helix formation with an amino acid that does not disrupt α helix formation. 6. The modified membrane disruptive peptide of claim 5 wherein the proline at position 14 of the melittin peptide is replaced with an alanine or tryptophan. 7. The modified membrane disruptive peptide of any one of claims 2 to 4, wherein the membrane disruptive peptide is modified to form a substantially continuous α helix by removing an amino acid which stops α helix formation. 8. The modified membrane disruptive peptide of any one of the preceding claims, which has been modified to form a dimer. 9. The modified membrane disruptive peptide of claim 8, wherein the membrane disruptive peptide is modified by replacing an amino acid of the membrane disruptive peptide with an amino acid that forms a covalent bond directly via a disulphide bond or via a linking group, with an amino acid of another modified membrane disruptive peptide. 10. The modified membrane disruptive peptide of claim 8, wherein the membrane disruptive peptide is modified by adding an amino acid capable of forming a covalent bond directly via a disulphide bond or via a linking group with an amino acid of another modified membrane disruption peptide. 11. The modified membrane disruptive peptide of any one of claims 1 to 7, which has been modified to form a trimer. 12. The modified membrane disruptive peptide of any one of claims 1 to 7, which has been modified to form a tetramer. 13. The modified membrane disruptive peptide of claim 11 or claim 12, wherein the membrane disruptive peptide is modified by replacing two or more amino acids with two or more amino acids that form disulphide bonds directly or via a linking group with an amino acid of other modified membrane disruptive peptides to form a trimer or a tetramer. 14. The modified membrane disruptive peptide of claim 11 or claim 12, wherein the membrane disruptive peptide is modified by adding two or more amino acids that form a covalent bond directly via a disulphide bond or via a linking group with an amino acid of other membrane disruptive peptides to form a trimer or a tetramer. 15. A modified membrane disruptive peptide having the amino acid sequence CIGANLKNLTTGLAALISWIKRKRQQ. 16. The modified membrane disruptive peptide of any one of the preceding claims, which comprises a lipid . 17. The modified membrane disruptive peptide of any one of the preceding claims, which has an extended α helix. 18. The modified membrane disruptive peptide of any one of the preceding claims, which has basic amino acid residues added to the C-terminius end of the membrane disruptive peptide. 19. A functional homolog of the modified membrane disruptive peptide of any one of the preceding claims. 20. Use of the modified membrane disruptive peptide of any one of the preceding claims in a delivery complex to deliver a negatively charged polymer to a cell. 21. The use of claim 20, wherein the negatively charged polymer is a nucleic acid. 22. A delivery complex for delivering a nucleic acid to a cell comprising a negatively charged polymer and the modified membrane disruptive peptide of any one of claims 1 to 19. 23. The delivery complex of claim 22, wherein the negatively charged polymer is a nucleic acid. 24. A delivery complex comprising a nucleic acid, a nucleic acid condensing peptide and the modified membrane disruption peptide of any one of claims 1 to 19. 25. A method for forming the delivery complex of claim 24 comprising: 1. condensing a nucleic acid with a nucleic acid condensing peptide to form a condensed nucleic acid complex; and 2. coating the condensed nucleic acid complex with the modified membrane disruptive peptide of any one of claims 1 to 19. 26. The modified membrane disruptive peptide of any one of claims 1 to 19 for use in therapy. 27. Use of the modified membrane disruptive peptide of any one of claims 1 to 19 in the manufacture of a composition for the treatment of a genetic disorder. 28. A method of treating a genetic disorder comprising administering to a patient in need of such treatment an effective dose of a delivery complex comprising a therapeutic nucleic acid and the modified membrane disruptive peptide of any one of claims 1 to 19.

Inventors: HUSAIN RHONDA DARPHI, PHILLIPS ROSS OWEN, WELSH JOHN HAMILTON, HUSAIN, RHONDA, DARPHI, PHILLIPS, ROSS, OWEN, WELSH, JOHN, HAMILTON
Applicants: COBRA THERAPEUTICS LTD, HUSAIN RHONDA DARPHI, PHILLIPS ROSS OWEN, WELSH JOHN HAMILTON, COBRA THERAPEUTICS LIMITED, HUSAIN, RHONDA, DARPHI, PHILLIPS, ROSS, OWEN, WELSH, JOHN, HAMILTON
Application year: 2000
Main classification: C07K14
IPC codes: A61K38, A61K47, A61K48, C07K14, C12N15