Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
  • C07K14/445—Plasmodium
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the field of the present disclosure relates to hybrid therapeutic peptides having the property of lowering the levels of free Tumour Necrosis Factor ⁇ and ⁇ in the circulation and other harmful cytokines, thus, modifying the pathological damage caused by Tumour Necrosis Factor, and finding a use in the treatment of diseases especially but not only septic shock; bacterial meningitis' cerebral malaria, HIV, SVHD graft versus host disease and pulmonary fibrosis.
• the present disclosure is an extension the teachings of PCT 93/00505 Anti viral fusion peptides whose teachings are incorporated herein fully by reference, which PCT was published as WO93/18160 after the filing date of the priority document GB93 19350.7.
• TNF Tumour necrosis factor
• TNF ⁇ Tumour necrosis factor
• TNF ⁇ Tumour necrosis factor
• Both ⁇ and ⁇ varieties have similar spectra of biological effects, however, TNF ⁇ is byfar the more powerful of the two.
• the effects of TNF are mediated by binding to specific receptors found on the surface of most cells.
• TNF has important biological roles to fulfil.
• Physiologically TNF is classified as a cytokine. It's effects on the immune system are to increase neutrophylia and activate macrophages, and increase the production of T cells in the thymus.
• TNF tumour necroses factor was attributed to this peptide by Old arising from the observation that TNF can reduce necrosis and regression of experimental sarcomas in mice.
• TNF serves to eliminate small tumours from the host at an early stage in their development.
• Clinically cloned TNF has been used to treat human tumours especially melanoma. It has also been shown that TNF is more effective against tumours when used in combination with other cytokines IL2 or ⁇ lFN gamma interferon.
• TNF microembili and haemorrhagic necrosis are produced in the substrate of the tumour leading to it's regression.
• this endogenously produced potent peptide is not always helpful to the host and may be harmful.
• Gram negative cell wall lipopoiysaccaride is a very potent agent capable of inducing a shock syndrome in low doses.
• Humans are extraordinarly sensitive to LPS.
• LPS has 3 components: Lipid A - R core - polysaccharide. The polysaccharide part varies with the species and strain of the infecting organism. The active moiety of endotoxin is now believed to be Lipid A.
• TNF may produce a significant part of the pathological spectrum of AIDS.
• Staal et al were able to demonstrate that intracellular thiols, more particularly GSH (gamma-glulamylcysteinglycine the most abundant), suppress NF-KB production and mitigate the effects of TNF ⁇ levels are elevated.
• Staal F Proc. Nalt. Acad. Sci. Usa, Vol 87, pp 9943-9947, Dec 1990.
• TNF ⁇ By blocking TNF ⁇ in vitro using anti-TNF ⁇ antibodies, the HIV production induced by TNF ⁇ can be reduced. Moreover, observers now suspect an amplification mechanism whereby TNF ⁇ leads to further production of it's own receptor and also production of IL6 (interleukin 6) which interleukin may prolong the process and continue to stimulate viral production when TNF is no longer available. Poli G et al, Proc. Nalt. Acad. Sci. USA, Vol 87, pp 782- 785, Jan 90.
• IL6 interleukin 6
• the agent methylxanthine pentoxyfylline is known to suppress TNF ⁇ levels in vivo. In vitro this agent has been demonstrated to reduce the replication of HIV in cultured cells. It's use in AIDS treatment in conjunction with the agent AZT was suggested by F Fazely et al, Blood, Vol 77, No 8, April 15 1991: pp 1653-1656.
• TNF ⁇ is known to suppress haematopoiesis, of itself, and via IL2 interleukin 2, levels of which are raised by TNF ⁇ .
• TNF ⁇ is a powerful suppressor of red cell production and this too is elevated in AIDS, as reviewed by J Doweiko in AIDS 1993, 7; 753-757. Anaemia is present in 35 to 75% of AIDS patients. It is difficult to treat and forces clinicians to lessen the dose of AZT or abort treatment with anti-viral agents.
• TNF ⁇ and ⁇ binds with high affinity to 2 different receptors a 55 kilo-Dalton receptor and a 75 kilo-Dalton receptor. Each receptor produces different pharmacological affects thereby broadening the range of activity of TNF. Significantly there are no TNF receptors on red cells. Indeed, the red cell is an exceptional cell in this respect.
• the teachings of the present disclosure enable TNF to bind harmlessly to red cells, thereby preventing it's deleterious effects.
• the 55 kilo-Dalton TNF receptor has been cloned and sequenced and one is directed to Loetscher H, Pan Y-C E, et al, Cell 61, (1990) 351-359 and also directed to Schall T J et al, Cell 61, (1990) 361-370, incorporated fully herein by reference.
• the 75 kilo-Dalton receptor has been cloned and sequenced and one is directed to Smith C A et al (1990) Science 248, 1019-1023 and to Dembic Z et al, 1990, Cytokine 2, 231-237, incorporated fully herein by reference.
• TNF binding proteins found in the blood stream are now believed to be free TNF receptors shed from cell membranes.
• such natural 'soluble' receptors are believed to control or modify some of the ill effects produced by TNF as suggested by Dan Aderka et al, J. Exp. Med., Vol 175, Feb 92, pp 323-329, and by Endelmann H et al, J. Biol.
• TNF-R55 and TNF-R75 show the receptors to be homogenous and belong to a wider family of similar receptors including NGF-R nerve growth factor receptor, CD40 and CD27, see Peter Vanderbee etal, J. Exp. Med., Vol 176, Oct 1992, 1015-1024.
• TNF-R(s) soluble TNF receptors To lessen the effects of TNF and develop anti-septic shock agents, some workers have tried to administer TNF-R(s) soluble TNF receptors.
• TNF-R the affinity of the immunoadhesion agent for TNF is improved. It's chemical efficacy likely to be greater by virtue of the fact that TNF a trimer is normally twice bound to it's receptor therefore preventing dimer adhesion to cell surface receptors which can still occur in the case of singly inhibited TNF molecules.
• the present invention teaches molecules or molecular machines having an affinity for TNF and human red cells. Thus enabling cells to mop up TNF and causing TNF to adhere to the red cell surface.
• the disclosure emphasises that TNF is not associated with red cells as a natural phenomenon.
• the present disclosure teaches an unnatural union between red cells and TNF, by means of novel pharmaceutical protein agents.
• the red cell surface protects the novel agents from excretion by the kidney.
• the red cell provides steric hinderance preventing a TNF so bound to itself from binding to a TNF-R in another cell.
• the present invention derives from PCT 93/00505 and teaches a cytokine receptor, fused to a malaria parasite peptide having affinity for a red cell, or analogue there of, and this provides a molecular machine, a hybrid fusion peptide capable of binding a cytokine (TNF ⁇ or ⁇ or interferons or interleukins) to the red cell surface thereby inactivating it.
• a cytokine TNF ⁇ or ⁇ or interferons or interleukins
• the present disclosure provides the advantage of a macromolecule capable of dual function TNF binding and red cell adhesion without any separate laboratory procedures on red cells being required.
• the present disclosure is not confined to TNF or its receptors but teaches the fusion of other cytokine receptors to malaria parasite peptides to produce macromolecules capable of reducing levels of harmful cytokines.
• novel macromelecular agents of the present disclosure bind directly onto red cells in one step, and provide a novel use for modified peptides of the malaria parasite organism.
• the malaria parasite has evolved from earliest times and attacks not only humans but most varieties of animal. This serious parasite infests red blood cells.
• Various malaria species infect humans, plasmodium faciparium, and plasmodium vivax being the most important.
• the life cycle is complex with a short life cycle in the salivary gland of mosquitoes and following inoculation of a human the parasite object is ultimately the red cell.
• Merozotes bind to the red cell membrane, enter the cytoplasm and multiply.
• the course of malaria is a variable one and may be characterised by a short acute illness which can bring death in a matter of hours; or a longer more chronic illness associated with debility and anaemia.
• malaria Other forms of malaria such as the plasmodium Knowlesi are well researched animal parasites which infects the Rhesus monkey.
• the preferred location forthe malaria parasite is within the red cells of the infected host and for much of its life span it lives intracellularly protected from the host immune system.
• glycophorin A molecule is a highly glycosylated peptide. Pasvol has suggested that glycophorin binding peptide binds to the region of glycophorin close to the lipid bi-iayer.
• glycophorin binding protein For several years a peptide called the glycophorin binding protein was believed to be the primary peptide responsible for binding merozoites to erythrocytes.
• a gene coding for GBP was isolated by M Ravetch J and Kochian J and disdosed in Science Vol 227, pp 1953- 1596, 29 March 1985 and incorporated herein fully by reference.
• GBP 130 is characterised by a tandem repeated sequence coding for a 50 amino add repeating sequence believed to be the site of erythrocyte binding, "A tandem repeated sequence determines the binding domain for an erythrocyte receptor binding protein of plasmodian faldparum". Cell, Vol 44, 689-696, March 14, 1986, Kochan J, Perkins M and Ravetch J. See Figure 2, p691, which also disdoses the full sequence and genetic code of the GBP 130 molecule.
• the EBA 175 molecule like the GBP 130 molecule has an affinity for the red blood cell surface and binds thereto.
• the EBA 175 molecule has a prediction for olygosaccharides which are found on the surface of the red cell molecule.
• nucleotide sequence of one form of the peptide GPBH is disdosed by Dagmar Nolte et al in the Journal of Molecular and Biochemical Parasitology, 49, (1991), p 253-264. See Figure 2 of p 257 incorporated herein fully by reference. The peptide sequence is also disclosed.
• Erythrocyte binding using different peptides and surface molecules is exhibited by other species of the malaria parasite in particularthe plasmodium vivax organism. This organism can infect only persons expressing the Duffy marker.
• the Duffy antigen is a red cell surface marker and is one of many blood group markers and is carried by a percentage of the population.
• Duffy antigens are therefore immune from infection by plasi ⁇ iQdium vivax.
• the plasmodium vivax expresses a Duffy binding receptor molecule P. vivax Duffy receptor was doned and sequenced by Xiangdang Fang and disdosed in Molecular and Biochemical Parasitology, 44(1991) p125-132. See especially Figure 1 of p127 for the genetic sequence and amino add sequence.
• plasmodium Knowiesi Similar to plasmodium vivax is plasmodium Knowiesi which also uses the Duffy antigen. This organism parasitises Rhesus monkeys. Also in the same Journal, same figure, same page, is listed the genetic sequence of plasmodium Knowiesi Duffy receptor molecule which may find a use in the agents of the present disclosure.
• malaria peptides When developing therapeutic agents directed against the malaria parasite itself, then it is clearly important to identify the precise molecule responsible for merozoite binding in the clinical context. However, where malaria peptides are to be employed as erythrocyte binding agents more generally, then it is not important to identify the precise peptide the malaria organism uses to effect invasion. Any malaria peptide capable of binding to an erythrocyte surface membrane may have a therapeutic use for other purposes such as the agents of the present disdosure and also segments of such a peptide.
• the present disdosure provides novel hybrid or fusion peptides having a minimum of 2 different peptide components each possessing different functionality.
• One peptide component will be derived from the malaria parasite or derivative or fragment or variation thereof and possess the ability to bind to a red ceil surface.
• the other component of the fusion peptide will be a cytokine receptor or derivative or fragment thereof especially the TNF-R75 and 55 (Tumour Necrosis Factor Receptor 75 kilo-Dalton and 55 kilo-Dalton, the IL1-R (interleukin-1 -receptor); the IL6-R (the interleukin 6-receptor); the IL8-R (the interleukin 8 receptor); IL2-R; IL4-R; IL3-R; IL5-R; IL7-R or the LIFR (leukaemia inhibitory factor)-R receptor or the ⁇ lFN-R gamma interferon receptor. It is envisaged that the fusion peptide will bind the target peptide to the red cell surface and mop up the free circulating target peptide (cytokine) thereby redudng it's deleterious effects. 14
• all or part of the peptide sequence comprising amino acid residues 201- 774 of the GBP 130 (glycophorin binding peptide molecule 130) all or part or substitutional or deletional variations thereof or fragments thereof especially tandem repeats or modified fragments thereof are modified to form hybrid fusion peptide drugs as shown herein below.
• Ctk-R represents a cytokine either TNF-R, IL1-R, IL2-R, IL4- R, IL8-R, IL6-R, IL3-R, IL5-R, IL7-R, LIF-R or ⁇ lFN-R or fragment thereof and where P represents the GBP130 molecule or fragment thereof, or other malaria derived red cell binding peptide.
• Exemplary embodiment 1(a). 1 is directed to a hybrid protein or fusion peptide capable of binding free TNF ⁇ or ⁇ to the red cell surface and comprising the fusion of two peptide components - a peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan etal in Cell, Vol 44, 689-696, Mar 141986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably, or by linkers to,
• Exemplary embodiment 1 (a).2 is directed to a hybrid protein or fusion peptide capable of binding free TNF ⁇ or ⁇ to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disdosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• Exemplary embodiment 1(a). 3 is directed to a hybrid protein or fusion peptide capable of binding free TNF ⁇ or ⁇ to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 141986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• Exemplary embodiment 1(b). 1 is directed to a hybrid protein or fusion peptide capable of binding free ⁇ lFN gamma interferon to the red cell surface and comprising the fusion of two peptide components
• - a peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 141986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to, - a peptide component derived from all or part of the ⁇ lFN receptor molecule the amino add sequence of which was deduced and cloned by Auguet M et al and disclosed in Cell 55(1988), 273-280 and incorporated herein fully by reference.
• Exemplary embodiment 1(b). 2 is directed to a hybrid protein or fusion peptide capable of binding free ⁇ lFN gamma interferon to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan etal in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• the agents of type example 1(b). 1 and 1(b). 2 may be especially useful in the treatment of G.V.H.D. graft versus host disease.
• Exemplary embodiment 1(c). 1 is directed to a hybrid protein or fusion peptide capable of binding free IL2 interleukin 2 to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally 18 to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• IL2 ⁇ a peptide component derived from the high affinity IL2 receptor ie. IL2 ⁇ as characterized by Bich-Thuy et al 1987, J. Immunology, 139(5), 1550-1556; Dukovich M et al, Nature (London) 327, 518-522; Hatakeyama M etal, 1989, Science 244; 551-556; Robb R J etal, Proc. Natl. Acad. Sci. USA, 84(7) 2002-2006; Saragon H etal, 1990, Proc. Natl. Acad. Sci. USA, 87 (1), 11-15; T Sudo M et al, Proc. Natl. Acad. Schi. USA, 84 (12), 9215-9218 disclosed and incorporated herein fully by reference.
• the agents of exemplary embodiment 1(c). 1 may be especially useful to reduce the expression of HIV virus in persons with AIDS.
• Exemplary embodiment 1(d). 1 is directed to a hybrid protein or fusion peptide capable of binding free II-6 interleukin 6 to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• peptide component comprising or derived from either the high or low affinity IL6 receptor as cloned by Yamasaki K et al, Science (1988), 241 , ⁇ 825-828 and described also by Taga T et al, 1989, Cell 58 (3), 573-581 ; and disdosed and incorporated herein fully by reference.
• Exemplary embodiment 1(e). 1 is directed to a hybrid protein or fusion peptide capable of binding free IL1 interleukin one to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• a peptide component comprising all or part or derived from the IL-1 R interleukin one receptor as disclosed by Sims J E et al (1988), Science 241, 585-589 and disdosed and incorporated herein fully by reference.
• One is also directed to C J McMahon et al to EMBO Journal Vol 10; No 10' 1991; pp 2821-2832 for details and sequence of a type II IL-1 receptor disdosed and incorporated fully by reference.
• Exemplary embodiment 1(f). 1 is directed to a hybrid protein or fusion peptide capable of binding free LIF leukaemia inhibitory fador to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• Exemplary embodiment 1(g). 1 is directed to a hybrid protein or fusion peptide capable of binding free Interleukin 3 to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan et al in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• Exemplary embodiment 1(h). 1 is directed to a hybrid protein or fusion peptide capable of binding free Interleukin 5 to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan etal in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N terminal end by peptide bonds preferably or by linkers to,
• Exemplary embodiment 1(i). 1 is directed to a hybrid protein or fusion peptide capable of 21 binding free Interleukin 8 to the red cell surface and comprising the fusion of two peptide components
• peptide component derived from all or part of the GBP 130 molecule (glycophorin binding peptide molecule as disclosed by Jarema Kochan etal in Cell, Vol 44, 689-696, Mar 14 1986, see Figure 2, p 691 and incorporated fully herein by reference fused N terminally to the C terminal end or C terminally to the N ten inal end by peptide bonds preferably or by linkers to,
• Exemplary Embodiment 1(j) is directed to a fusion peptide of a malaria parasite red blood cell binding peptide such as GBP130 fused to the IL4R interleukin 4 receptor all or part.
• the IL4 receptor was cloned and disclosed by R L Idzerda et al, J, Exp. Med., Vol 171 , Mar. 1990, pp861-873, incorporated fully herein by reference.
• the agents of group 2 use the Glycophorin binding peptide homologue molecule for red cell binding.
• glycophorin binding peptide homologue molecule was cloned and disclosed by Dagmar Nolte et al Molecular and Biochem Parasitology, 49(1991) page 253-264. See especially Figure 2, p257 and is incorporated fully herein by reference.
• the exemplified agents of group 2 are identical in every respect to group 1 except that in place of GBP 130 or segments thereof the malaria RBC binding component is provided by 22 GBPH glycophorin binding peptide homologue as referenced herein above especially peptide fragments comprising amino add residues: residue 70 to 427 inclusive residue 109 to 427 inclusive residue 230 to 268 or any other tandem repeat or polymer thereof of any fragment or species variation or substitution or deletional or inclusional variant thereof.
• the agents of group 3 use the EBA 175 erythrocyte binding antigen 175 for red cell binding.
• the EBA 175 erythrocyte binding antigen 175 was doned and disdosed by B Kim Lee Sim et al Journal Cell Biology, Vol. Ill, 1990, p1877-1884. See especially Figure 2, p1880 and is incorporated fully herein by reference.
• the exemplified agents of group 3 are identical in every respect to group 1 except that in place of GBP 130 or segments thereof the malaria RBC binding component is provided EBA 175 erythrocyte binding antigen 175 espedally peptide fragments comprising amino add residues i residue 20 to 1435 indusive of any other fragment orspedes variation or substitution ordeletional orindusional variant thereof.
• the agents of group 4 use the Plasmodium Vivax Duffy Receptor for red cell binding.
• Plasmodium Vivax Duffy receptor was doned and disdosed by Xiangdong Fong et al Molecular Biochemical Parasitology, 44 (1991 ) 125-132. See especially Figure 2, p 127 and is incorporated fully herein by reference.
• the exemplified agents of group 4 are identical in every resped to group 1 except that in place of GBP 130 or segments thereof the malaria RBC binding component is provided by
• malaria parasite derived component is represented all or in part by i an anti-ideotype Fab fragment ii an antibody fragment binding to red cells in the same way as the malaria parasite components.
• the protein of the present disdosure are fabricated preferably in a stepwise fashion. Many different manufaduring strategies are available for each component any or all of which may 24 be applied in various combinations didated largely by two fadors.
• Newer techniques for multicomponent peptide synthesis permit the simultaneous synthesis of oligo peptide segments in a single run thus redudng time and costs considerably.
• Detachment of the peptide from the HYCRAM® support employs palladium tatra-kis (triphynyl-phosphane) a catalyst in a suitable solvent such as 50% (v/v) dimethylsulphoxide 26 with dimethyl formamide' N-methyppyrrolidine, tetrahydrofuran and water. Oxygen tetrahydrofuran must be exduded. Acceptor molecules, morpholine, dimedine or N, N'- dimethylbarbiturate may be added to take up the allylic group.
• the Ddz-/t-butly amino acid protedions are easier to cleave using with 1-5% (v/v) trifluroacetic acid in dichloromethane a process taking 10 to 30 minutes or by means of the more environmental friendly acetic add or dioxane containing 1% (w/v) HCL gas.
• the other useful protocol is the Fmoc-/t-butyl strategy. Cleavage of F moc can be achieved using 20-50% (v/v) piperidine/dimethyl formamide.
• Deprotedion can be monitored in both cases photometrically.
• the adivation of Boc-; Fmoc-; orDdz-aminoacid derivatives may employ the inexpensive (Dccdidohexylcarbodi- imide.
• Pre activation using HOBT (N-hydroxybenzotriazole) can be employed to form symmetric anhydrides of proteded amino adds or their esters.
• activating agents are the Castro Reagent or BOP' Benzotriazole-1-yl-oxy-tris (dimethyl amino) phosphonium hexa flurosphosphate; one is direded to CASTRO b et al (1957) Tetrahedron Lett. 15, 1219; and TBTU the Knorr reagent, Benzotriazole-1-yl-oxy-1, 1, 3-tetramethyluronioum tetrafluoroborate one is direded to Knorr R et al (1989) Tetrahedron Lett. 30, 1927.
• Fragment condensation can be achieved using the BOP or the TBTU reagent with HOBT in excess. Proteded peptides must also be in excess, however, solvents and excesses can often be recycled.
• Atypical produdion process involve either the separate synthesis of peptide sequences by their expression in suitable hosts, and their subsequent purification; or chemical synthesis such as on a solid substrate for example by the sequential addition of amion add residues or peptide fragments which are proteded, the protedion of the amino add residues as required and the subsequent reading of the peptide chains with linking agents before removing the peptide chains from the said solid substrates and the final purification by the various means is such as reverse phase chromatography; or any combination of the above.
• a fused gene is a genetic sequence which codes both components of the hybrid component molecule.
• a fused gene is a genetic sequence which codes both components of the hybrid component molecule.
• peptide fragments may be manufadured by DNA cloning and expression in suitable hosts and recovery with subsequent condensation in vitro.
• cloned sequences useful for the produdion of fusion peptides will have the transmembrane domain and the cytoplasmic domain sequence removed.
• DNA may be made by the chemical synthesis of DNA polymer fragments using phosphotriester, phosphite or phosphoramidite chemistry.
• phosphotriester, phosphite or phosphoramidite chemistry For a description of sold phase techniques one is direded to Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory manual ed H G Gassen and L Lang, Verlag Chemiee, Weinheim 1982; and Gait M J et al Nucleic Acids Research 1982, 10, 6243; Spoat B S et al Tetrahedron Letters, 1980, 21, 719; Matteuci M D et al J. American Chemical Society, 1983, 195, 661; Sinha N D et al Nucleic Acids Research, 1984, 12, 4539 and Matthes H W D et al Embo. Journal, 1984, 3, 801, whose teachings are incorporated herein fully by reference.
• Reverse transcriptase techniques may also be used to generate a complimentary cDNA strand by means of the reverse transcription of malaria parasite derived mRNA. Kits are available for this purpose.
• the DNA fragments maybe ligated by either blunt-ended orstaggered-ended termini after using restridion enzymes; digestion; filling in a required; and treatment with alkali and phosphatase for protedion and subsequent ligation with suitable ligases.
• leader sequences may be chosen from the many available.
• the cloning of the DNA sequence of the hybrid peptides of this invention may take place in prokaryotes such as E.Coli for example, K12 strain or E Coli B by way of non limiting examples or by means of the polymerase chain reaction.
• hybrid peptides may take place in any host cell, induding mammalian host cells.
• Other useful cells are fungi, yeasts, inseds and prokaryotes.
• Signals suited to the chosen host cell are chosen as appropriate, in the case of prokaryotes one can chose from a large group induding alkaline phosphatase, pemallinase and the like.
• prokaryotes such as E Coli for example, are used to express the hybrid peptides, then they are transformed by an expression vedor usually a plasmid sudi as PBR322 into which the DNA encoding the fusion peptide or fragment has been ligated such as plasmid will also feature suitable marker sequences, promoters, and Shine-Dalgarno sequences may be chosen as appropriate.
• a prokaryote host such as E Coli may be transformed by treatment using a solution of CaCI 2 as described by Cohen et al PNAS 1973, £2, 2110 or by treatment with a solution comprising a mixture of RbCI, MnCI 2 , potassium acetate and glycerol and then subsequently with 3 -[N-morpholino] - propene-sulphonic add and, RbCI and glycerol.
• a solution of CaCI 2 as described by Cohen et al PNAS 1973, £2, 2110
• a solution comprising a mixture of RbCI, MnCI 2 , potassium acetate and glycerol
• 3 -[N-morpholino] - propene-sulphonic add and, RbCI and glycerol.
• a suitable vedor would be Bacuiovirus.
• Bacuiovirus Such a system would contain the target peptide encoding sequence linked to a bacuiovirus promoter within a shuttle vedor with sufficient bacuiovirus DNA flanking the target peptide encoding sequence to permit recombination.
• One is also direded to Smithklein (WO/US/89/05550).
• Insed larvae can also be direded to PCT/WO/88/0200030 Miller et al.
• Other useful inseds are Drosophila melanogasteric, and the like.
• cowpea plant provides a suitable expression system.
• CPMV cowpea mosaic virus
• a general protocol for the cloning of foreign genes in plants (tomatoe) and the like may be obtained by consulting HORSC R B et al Science 227, 1229- 31.
• the plasmid YRp7 as the expression vedor may be used.
• the chosen host is a yeast such as Saccharomyces cerevisiae
• the plasmid YRp7 as the expression vedor may be used.
• promoters A wide choice of promoters is available for use in yeast cell expression systems and indude byway on non limiting examples 3-phosphoglycerate kinase and one is direded to Hitzman et al J. Biol. Chem. 255 2073 (1980); also enolase, glyceraldehyde-3-phosphate dehydroginase, hexokinase, pyruvate decarboxylase, phosphofrudokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phospho glucose isomerase and glucokinaise being glycolytic enzymes and one is direded to Hess et al J. Adv. Enzyme Reg. 7, 149 (1968) and to Holland Biochemistry 17, 4900 91978.
• promoters suitable for yeast expression systems indude the promoter regions for alcohol dehydrogenase 2, 100 cytochrome C, acid phosphatase, also mettallothioneins, glycoraldehyde-3-phosphate dehydrogenase and others one is direded to Hitzman R et al European patent Publication No. 73, 657A.
• mammalian cells are the chosen hosts for expression, these cells may be grown in vitro in tissue culture or suitable bioreadors or in vivo in animals.
• Vedors useful for mammalian cells host systems involve the use of DNA derived from animal viruses such as SV40 virus; retroviruses such as RSV, MMTV, MOMLV, 31 bacuiovirus, Vaccinovirus, Andeovirus, polyoma or bovine papilloma virus.
• animal viruses such as SV40 virus
• retroviruses such as RSV, MMTV, MOMLV, 31 bacuiovirus, Vaccinovirus, Andeovirus, polyoma or bovine papilloma virus.
• Promoters suitable for mammalian cells systems may be chosen from the many available.
• Some techniques useful for the introdudion of the expression vedor into the host cell involve protoplast fusion, calcium phosphate precipitation, eledroporation, and other techniques.
• Mammalian host cells suitable for the expression of fragments of or in some cases the entire fusion peptides may now be chosen from the large number now available such as VERO or CHO-K1, a myloma cell line.
• eukaryotic host cells indude COS cells, human embryonic kidney cells, mouse plasmoacytoma cells, mouse sertoli cells, baby hamster kidney cells (BHK cells), Chinese hamster ovary cells - DKFR(CHO), monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells (HELA cells), canine kidney cells, Buffalo rat liver cells, human lung cells, human liver ceils and mouse mammary tumour cells, byway 32 of non limiting examples.
• the chosen host cells will preferably express the minimum levels proteases within their cytoplasm. It will also be appreciated that amino add sequence variations of the peptide sequences involved may be insertions, substitutional or deletional variations involving single amino add residues or peptide fragments.
• the purpose of such variations may be to increase the affinity of the components, to improve stability, to reduce the cost preparation or to increase the half life, or to lessen the severity of side effeds sudi as atopic readions.
• the final form of the agents of the present disclosures may involve any combination of substitutions, deletion or insertion of amino add sequences, provided the binding ability of the epitopes or peptide sequence are retained.
• glycosylation variations ie. variants completely unglycolated, variants having glycosylta ⁇ d amino adds other than those glycosylated in the natural peptides or variants having a greater number of amino adds residues glycosylated or fewer glycosylated residues or residues glycolated by oiigosaccharides other than those oligosaccharides usually associated with the said sequences.
• the peptides are recovered and purified by means known to the ordinary skilled artisan.
• Such methods may include add extradion, ethanol precipitation using ammonium sulphate, anion or cation exchange chromatography phosphocellulose chromatography, immunoaffinity, chromatography hydroxyappetite chromatography and reverse phase chromatography.
• purification and processing involves four stages:
• Extradion may be accomplished using sonication techniques or solid shear techniques - on a small scale.
• Lysozyme based techniques are expensive. More usually in the case of baderial hosts homogenizers or liquid shear techniques are employed.
• a produd is expressed as an indusion body cell paste may be solubilised by solvents such as 8M-guanidinium.
• Centrifugation provides the removal cellular debris, and continuous flow centrifuges are preferred for large scale operations.
• cross flow filtration using flat to tubular membranes and high shear forces may provide a useful alternative to centrifugation.
• Precipitation using ammonium sulphate, organic solvents, polyethylene glycol or other polymers can be used to accomplish this step; with the addition of some variety of chromatography usually absorptive chromatography techniques employing ion exchange, hydrophobic or bioaffinity interadions; followed by washing and desorption; chromatography is not restrided to columns but may take place in membranes or even in spirally wounded cartridges.
• a way around this problem is to oxidize disulphides under denaturing conditions, using gel filtration remove covalent aggregates and thereafter dilute the produd in a non denaturing buffer.
• the peptide which collapses into an amorphous tangle often rearranges itself into the native form.
• Ion exchange chromatography techniques are very useful in early purification stages and can deal with large volumes at great speed, producing yields of high resolution.
• Hydrophobic interadion chromatography provides both high resolution and high speed even at large batch volumes.
• Bio affinity chromatography produces the highest resolution, at high speed, but batch size may require curtailment. This technique is an ideal late stage technique.
• Particle size is decreasing from the 90 ⁇ m of traditional gels to approximately 40 ⁇ m for newer gels such as Sepharose HR®, Suphacryl HR®, Superdex® (Pharmacia - LKB) or Fradogel® (Toso-Haas).
• the process of chromatography involves the choice and development of a strategy containing one or more steps ie. high resolution single step or a multistep procedure the final choice to be determined by (1) the chosen peptide produdion process which determines the form of the starting material to be purified
• the first chromatography column will involve large diameter packings circa 100 ⁇ m, which are often chosen so that they can be resanitized by sodium hydroxide to reduce costs. Low resolution steps to be followed by higher resolution steps until the desired produd purity is obtained.
• Step Two Re-run through h.l.c column adding salts to bind the target peptide to the h.l.c column.
• the objedive is volume reduction.
• Alternatively employ ultra filtration as described. Use a step gradient to elute the target peptide.
• Step Three A step gradient to a low ionic strength buffer will remove remaining salts.
• Alternatively employ polyethyleneimine precipitation, centrifugation and diafiltration.
• Produd polishing involves the removal of polymers of the produd and other pyrogens.
• a sample of patients blood 50ml is colleded in a suitable container. It is desired to measure free TNF ⁇ or levels of circulating cytokines to determine elevation of these levels implying say endotoxic shock.
• 1mg of the hybrid fusion peptides of group 1a1, 1a2 or2a1 or 3a or 4a or 5a are mixed with 10ml of blood to be assayed. Thereafter the antibodies direded against the complex TNF- TNF-R-malaria parasite peptide are added. These antibodies may be radiolabelled or labelled with luminescent groups or labelled by other means.
• the blood sample is then spun and rediluted, re-spun and rediiuted. Thereafter the sample is put through an automated counter.
• the agents attach individual molecules of TNF to red cells and permit visualization and quantification of very small amounts, molecule by molecule of TNF on a red cell with very few laboratory steps. Moreover, as red cells lack TNF receptors the procedure should be accurate and permit measurement of very small amounts of TNF. As for TNF other cytokines may be assayed in the same way.
• cytokine receptor sequences can be listed and are excluded in the interest of brevity. Those cytokine receptor sequences not listed are well known to the skilled artisan and freely available in referenced texts and literature of the art. The substitution of such unlisted receptor sequences involves no intensive steps and falls within the skill of the ordinary skilled artisan.
• TNF-receptor malaria peptide fusion peptide A Examples of TNF-receptor malaria peptide fusion peptide.
• the TNF receptor sequence is in accordance with H Loetscher et al Cell, Vol.61, 351-359, April 20; 1990, p353, Fig.2A, the malaria parasite components as referenced herein before,
• H1 In this example the IL2-R molecule is in accordance with part of the IL2 molecule disdosed by T Nikaido et al, Nature, Vol 311, 18 Od 1984, pp631 to P.635 incorporated fully herein by reference. H1 t_ 2 - IL2-R - GBP 130 - COOH
• the IL2 receptor molecule is in accordance with part of that molecule as disdosed by D Cosman et al, Nature, Vol 312; 20/27, December 1984, pp768- 771 especially page 770 incorporated fully herein by reference.
• the IL2 receptor is formed by the IL2-R ⁇ chain as disdosed by Masanori Hatakeyama et al and one is direded to Science, Vol. 244, 5 May 1989, pp 551-556 see especially page 552 figure 1B.
• Example of IL-3R-GBP130 fusion protein the IL-3R amino acid sequence is disdosed by Toshio Kitamura et al Cell, Vol 60, 1165-1174, Sept 20, 1991 and incorporated fully herein by reference.
• TNF-receptor malaria peptide fusion peptide A5 Examples of TNF-receptor malaria peptide fusion peptide.
• the TNF receptor sequence is in accordance with H Loetscher et al Cell, Vol. 61, 351-359, April 20; 1990, p353, Fig.2A, the malaria parasite components as referenced herein before,
• A6 NH.-TNF-R - GBPH-COOH (glycophorin binding peptide homologue)
• An example of a polymer of an amino acid repeat sequence NH 2 - He Tyr Pro Ser Gly Val He Gly Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg
• n a real number

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